Download molecular biology

MOLECULAR BIOLOGY
Translation
Kolluru. V. A. Ramaiah
Professor
Department of Biochemistry
University of Hyderabad
(Revised 30-Oct-2007)
CONTENTS
Introduction
Messenger RNA (mRNA)
Splicing
Addition of 5’Cap
Addition of poly A tail
RNA editing
Ribosomes
Subunits, composition, morphology
Processing of rRNA
Functions of ribosomal subunits
Polysomes
Transfer RNA (tRNA)
Processing
Modified bases
Charging or aminoacylation
Genetic Code
Cell-free translational systems
Synthetic mRNA templates
Amino acid analyses of polypeptides produced by synthetic templates
Codon- charged tRNA- ribosome complexes
Wobble and degeneracy
Mutations
Gene density and overlapping genes
Protein synthesis
Initiation
Elongation
Termination
Ribosome recycling
Differences in the initiation in eukaryotes and prokaryotes
Inhibitors
Translational regulation
Different RNAs and functions
Co- and Post-translational Modifications of proteins
Keywords
Messenger RNA (mRNA); Ribosomes; Polysomes; Transfer RNA (tRNA); Aminoacylation; Genetic code;
Translational system; Wobble; Mutations; Gene density; Protein synthesis; Translational regulation.
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Introduction
Proteins are biological polymers like the nucleic acids. The alphabets of protein language are
however different from that of nucleic acids. The monomeric units of proteins are the alphaamino acids. The twenty naturally occurring amino acids are like the alphabets of a language that
go into the composition of different proteins. A typical α-amino acid consists of an amino group
(-NH2), carboxyl group (-COOH) and an R group. It is the R group that gives specificity to an
amino acid. The carboxyl group is attached to the α-carbon, the carbon next to the carboxyl
group (Fig. 1). Amino acids in a protein chain are linked by a peptide bond (
). Usually,
proteins have an N-terminal end carrying a free amino group and C-terminus with –COOH. The
linear sequence of amino acids in each protein is specific like the alphabets in a word. Proteins
are vital for life and mediate a variety of functions: in storage, structure, catalysis, signaling,
transport, defense, transporting ions, blood clotting, and muscle contraction (Fig.2). Proteins like
prions even mediate transmissible diseases called spongiform encephalopathies and the structure
of a protein determines its function. Hence the study of biological synthesis of proteins and their
modifications to attain a proper 3-dimensional structure are important. Approximately 35-45%
genes make products devoted for translation, and 35-40% of the total energy generated, for
example by E. coli, is consumed for protein synthesis. Hence the synthetic process and also the
modification process require close monitoring and regulation.
The information for the synthesis of a protein is stored in the nucleotide sequence of messenger
RNA (mRNA) that in turn is synthesized from the corresponding DNA. The process of RNA
synthesis from a DNA template is called transcription and the synthesis of proteins from an
mRNA template is called translation. The information flow also occurs from an RNA template
to a DNA molecule through a process called reverse transcription where DNA is synthesized
from an RNA transcript in the presence of an enzyme called reverse transcriptase (Fig. 3). In
prokaryotes that lack defined organelles or nucleus, the process of transcription and translation
are coupled whereas in eukaryotes, the RNA synthesis occurs in the nucleus and the protein
synthesis takes place in the cytosol, organelles, or on the surface of endoplasmic reticulum (ER).
Organelle protein synthesis resembles prokaryotic protein synthesis. Proteins made on the
surface of ER membrane are called secretory proteins (eg: serum albumin, immunoglobulins,
digestive enzymes, egg white proteins etc.,) that are destined to reach various
locations/organelles in the cell. Secretory/membrane proteins carry an N-terminal signal
sequence that is rich in hydrophobic amino acids. Such a signal sequence is absent in cytosolic
proteins. The rate of translation of eukaryotic mRNAs is slower than in prokaryotes because of
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the spatial and temporal separation of transcription and translational events, processing of the
premessenger RNAs (mRNAs) that remove the non-coding regions, and their ability to undergo
distinct posttranscriptional modifications at the 5’ and 3’ends. Several complex regulatory
mechanisms control the gene expression in eukaryotes and they occur at different levels:
transcription, post transcription, transport of RNAs into the cytosol, translation, and the
degradation or stability of mRNA.
Like the synthesis of DNA (replication) and RNA (transcription), specific cellular machinery
directs protein synthesis (translation). While differences exist, one can also see some common
pattern directing the biological synthesis of these polymers viz., proteins, RNA and DNA. The
syntheses of all these molecules require a template strand that contains the necessary information
with ‘start’ and ‘stop’ signals. The addition of monomers occurs on the template. These
monomers come one after another based on the sequence information coded in the template and
attach to the template molecule. A covalent bond like phosphodiester bond joins the adjacent
nucleotides in DNA and RNA synthesis, whereas a peptide bond brings together adjacent amino
acids in protein synthesis. The primary products, or the newly made RNA and protein molecules
are processed where certain nucleotides or amino acids that are non-coding or non-essential for
function or structure are selectively removed to yield active molecules. Processing of RNAs and
pre-proproteins yield biologically active RNA and protein molecules that are devoid of ‘introns’
as in RNA, or prepro-amino acid sequences or ‘inteins’ as in proteins. In addition, eukaryotic
RNAs and the amino acids in proteins are also modified in several other ways during or after
their synthesis and these are called post-transcriptional or post-translational modifications
respectively that play important role in their functions (see latter).
The expression of genes i.e., the synthesis of RNA and proteins is regulated or controlled by a
wide variety of ways. While the DNA of a genome is replicated completely preceding the cell
division but only portions of DNA are transcribed and or translated at different periods in the
development or in different cell types. The genetic content between men and mice is 97.5%
similar and the 2.5% difference separates mice from men. Among organisms, like human beings,
the difference in the genetic content is 0.1% and that small difference distinguishes one
individual from the other for their appearance, behavior and predisposition to certain diseases.
Although different cell types like muscle cells, red blood cells and cells of pancreas differ in
their functions, their genetic content is 100% similar because they are all derived from the same
parent cell. Similarly a normal cell differs from an abnormal, or aged, stressed, diseased or virusinfected cell. An unfertilized egg differs from a fertilized egg. A dormant seed is different from
germinating seed. This is because the gene expression or the types of RNAs and proteins made
by these cells are different. Hence the gene expression critically regulates the development,
differentiation of cell types and plays a role in health and disease.
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In brief, the protein synthesis machinery consists of different types of RNAs (Box-1), ribosomes,
enzymes such as aminoacyl synthetases that catalyze the transfer of amino acids to
corresponding tRNAs, and a variety of protein factors that are required during the substeps in
initiation, elongation, termination of translation and in ribosome recycling. The process of
protein synthesis consumes a lot of ATP and GTP. While ATP hydrolysis is used in driving the
protein / peptide synthesis and in unwinding any secondary structures in mRNA, GTP hydrolysis
facilitates conformational changes in ribosomes that are required in the joining of small subunit
to the large ribosomal subunit, the translocation/movement of tRNA from one codon to another
and also in the detachment of the factors from ribosomes. Ribosomes, and other components of
protein synthesis that are involved in the decoding of information in mRNA are relatively stable
compared to the template mRNA. In addition to the three RNAs mentioned in Box-1, several
other RNAs with a variety of functions play a role in the synthesis of DNA, RNA and proteins
and also in controlling gene expression (see latter).
Messsenger RNA (mRNA)
One of the DNA strands serves as a template for the synthesis of messenger RNA (mRNA).
Messenger RNAs are protein-coding RNAs containing typically three regions: a 5’UTR
(untranslated region), a protein coding sequence (open reading frame or ORF) and 3’UTR (Fig.
4B). The protein coding sequence of mRNA has a ‘start’ site at the 5’side (ribosome binding site
or RBS and is about 7-10 nucleotides in length) and ‘stop’ site at the 3’end. Eukaryotic mRNAs
are mostly monocistronic coding for a single polypeptide whereas prokaryotic mRNAs are
polycistronic with multiple starts and stop signals and coding for more than one protein. In
prokaryotes that lack a defined nucleus, the process of RNA and protein syntheses are coupled.
This means, as and when the mRNA is being transcribed from a DNA template, the RNA
transcript is translated by ribosomes to the corresponding protein. Further, prokaryotic mRNAs
lack additional features such as a cap structure at the 5’ end and poly A tail at the 3’end which
are found in most eukaryotic mRNAs. In eukaryotes, the transcription or the synthesis of
mRNAs occurs in the nucleus. The pre-mRNAs are called heterogenous nuclear RNA (hnRNA).
The hnmRNA or pre-mRNA molecule is bound by a spliceosome that contains several proteins
and uracil-rich small nuclear RNA molecules. The primary transcripts of eukaryotes are
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unusually long and contain coding and non-coding sequences called exons and introns
respectively. Because of this reason, the eukaryotic genes are called split genes. This can be
demonstrated by imperfect hybridization of the DNA template with its mature or processed
mRNA transcript. Part of the regions in DNA (corresponding to introns) cannot be base paired
with its mature mRNA and form single stranded loops. This can be detected using an electron
micrscope or by using S1 nuclease that attacks preferentially the single stranded unpaired region
in DNA. The fragments of DNA generated by S1 nuclease digestion can be separated on an
acrylamide/ agarose gel electrophoresis. Thus the mature RNAs do not have certain sequences
that are complimentary to their template DNA molecules and these are called introns or noncoding sequences.
Splicing
Splicing or joining of the exons (Fig. 4A) and removal of the introns involve two successive
transestrification reactions in which the phosphodiester linkages within the mRNA are broken
and reformed as shown below (see figure). Introns are incidentally ancient and are found in
bacterial tRNAs, but not in their mRNAs. Archaebacteria too have introns both in their tRNAs
and in the ribosomal RNAs. Eukaryotes have introns in many of their pre-RNAs including
mRNAs. This suggests that probably they are lost in bacterial mRNAs due to evolutionary
constraints. An analysis of eukaryotic DNA sequences at the boundaries of exons and introns
revealed that introns mostly have GU sequence at the beginning (on their 5’ side) and AG
sequence at the end (on the 3’ side). In addition to these sequences, splicing of introns requires a
pyrimidine rich sequence called the polypyrimidine tract (U/C)11 preceding the AG nucleotides
at the 3’ side of intron and a branch point sequence consisting of 5’- YNYYRAY (Y represents
any pyrimidine, N refers to any nucleotide and R means a purine A or G, and A is an adenine)
that is present 10-40 residues upstream (that is towards the 5’end of the intron) to the above
polypyrimidine tract.
Fig. 4 A and B
Based on the mechanism of splicing, three groups of introns are identified. Removal of group I
introns, for example present in rRNAs, can proceed in the absence of any proteins. However, the
splicing reaction of group I introns require the presence of guanosine and Mg2+. The concept of
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RNA enzymes has come from such splicing reactions. The RNA enzymes like the protein
enzymes increase the rate of reaction by several fold and require typical 3-D structure of their
substrates. However unlike most protein enzymes, RNA enzymes act on themselves (selfsplicing) and are modified at the end of reaction. Essentially RNA enzymes have nuclease and
polymerase activities. They can remove nucleotides and can also add nucleotides through a
transesterification reaction. The removal of group II and III introns (premRNAs) are similar to
each other except that the removal of group III introns requires the participation of a
spliceosome, a complex of several RNAs and proteins. Unlike group I introns, the splicing of
group II and III introns does not require guanosine. In both cases, the OH-group of adenine
nucleotide that is part of the intron branch point sequence attacks the 3’end of the exon present at
the 5’end of the mRNA. Afterwards, the free 5’end of intron joins the adenine nucleotide in
intron to give a lariat structure, whereas the -OH group that is generated at the 3’end of exon will
attack the 3’end of intron thereby releasing the intron. At the same time, two exons present at the
5’ and 3’ends of the intron are joined with each other (Fig. 4C).
Alternative Splicing
Alternative Splicing produces multiple mRNAs that code for different proteins where as normal
splicing includes all exons and elimination of introns. The processing of pre-mRNAs is not
always uniform. Some times the processing facilitates the joining of different combinations of
exons of an mRNA. Also, the cleavage and polyadenylation of pre-mRNAs (Fig. 6) at different
sites in the 3’ end would produce different lengths of mRNAs. For example, splicing and
3’cleavage sites of the calcitonin mRNA that produces calcitonin hormone in the cells of thyroid
gland and brain differ to produce different proteins. The mature transcript of the calcitonin
mRNA is found long in brain cells than in thyroid cells. This is because the thyroid calcitonin
mRNA contains four of the six exons (1-4) without any introns. However in brain cells, the
cleavage and polyadenylation of pre-mRNA occurs at the end of sixth exon. The processed or
mature mRNA in brain cells has exons 1, 2, 3, 5 and 6 and does not have any of the introns.
Exon 4 is not included. Alternative splicing may explain the diversity of proteins produced in
eukaryotes that apparently do not correspond to their estimated number of functional genes. In
humans, for example, approximately 30,000-35,000 functional genes code for millions of
proteins.
The processing of heavy chain pre-mRNAs of IgM class immunoglobulins of B cells and plasma
cells, and the α-tropomysin gene in different muscle cells are some of the other examples of
alternative splicing. This may be possible because of the presence /absence of specific cellular
splicing factors. The plasma cell that secretes immunoglobulins into the blood produces a mature
IgM transcript that retains one of the exons which produces a stretch of hydrophilic amino acids
and is consistent with the ability of plasma cell to secrete the protein. In contrast, the B-cell
produces a mature mRNA transcript that encodes a stretch of hydrophobic amino acids that
facilitates the protein to be anchored in the plasma membrane. Analysis of some proteins like
LDL (low density lipoprotein) –receptor indicates that the various domains of this protein come
from different exons probably by a process called exon shuffling. These domains in LDL protein
are strikingly similar to other proteins like epidermal growth factor receptor, blood clotting
factors and C9 complement factor.
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Fig. 4C: Processing of different introns
Many of the examples cited above suggest that introns of pre-mRNAs contain non-coding
regions and are eliminated during splicing. The joining of different combinations of exons in a
pre-mRNA transcript produces proteins of different kinds. However this is not true always. DNA
sequences that become part of intron sequences of some pre-mRNAs are also shown to code for
proteins suggesting that genes are embedded in genes. Examples include the pupal cuticle gene
of Drosophila is an intron of another gene that codes an enzyme important in the synthesis of
purines (adenine and guanine). While the sequence is excluded in the mature RNA transcript of
the purine synthesizing enzyme, it however produces pupal cuticle mRNA of 0.9 kilobase when
it is transcribed separately from the DNA sequence.
Introns are longer than exons generally, and may a play a role in the transport of processed
mRNAs from nucleus to cytoplasm. Mutations in an intron can disrupt the correct splicing and
can enhance the transforming activity of certain oncogenes (ex: ras). Introns mark the functional
protein regions while exons encode well-defined structural domains in proteins.
Addition of 5’ Cap
Most of the eukaryotic mRNAs contain a cap structure consisting of 7-methyl guanine, an
additional nucleotide at the 5’end. The guanine nucleotide is added soon after the initiation of
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transcription (even before the completion of the transcription of the mRNA) and is a cotranscriptional event as illustrated (Fig. 5).
The guanine nucleotide joins the 5’-end of the
mRNA by a 5’-5’ bond instead of a 5’-3’ phosphodiester bond that joins all the other
nucleotides. Afterwards, a methyl transferase enzyme adds a methyl group at position 7 of the
newly added guanine nucleotide. Also the 2’ OH group of sugars joined to the second and third
nucleotides of mRNAs is methylated. The 5’ cap may offer stability to eukaryotic mRNAs and
regulates translation by providing a binding site for several factors.
Addition of Poly A–tail
In addition to the removal of introns, the pre-mRNAs in eukaryotes are processed, at a position
from 11-30 nucleotides downstream of an AAUAAA consensus sequence, in their 3’untranslated
regions. The cleavage is aided by polyadenylation specificity factor and cleavage stimulation
factor. These factors bind to the RNA polymerase during transcription when the polymerase
reaches the end of the transcribing gene. After the cleavage, a number of adenine nucleotides are
added by the enzyme polyA polymerase in the presence of ATP to the 3’ end of a cleaved
mRNA (Fig. 6). The presence of poly A tail may confer stability to mRNA and also helps in the
circularisation of mRNA during translation due to an interaction between proteins bound to the
5’and 3’ends of mRNA. The pseudo-circularisation process may facilitate an efficient reinitiation
of protein synthesis in eukaryotes (see latter).
Fig. 5: Steps in the addition of 5’ Cap in eukaryotic mRNAs
RNA editing
This is yet another way of modifying the transcribed RNA and is different from splicing. Here
nucleotides are added, deleted, or both events can happen. It can occur by chemical
modifications as in the conversion of cytosine to uracil by a process called deamination or by a
guide RNA. In the editing process, the RNA transcript is modified but not the DNA. For
example, apolipoprotein mRNA in some cells produces a full protein with a higher molecular
mass. However, the same mRNA has a stop codon in some cells and produces a truncated protein
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with a lower molecular mass, approximately half of full form of the protein. In the latter, one of
the mRNA codons, CAA, that codes for the amino acid glutamine is modified to UAA, a stop
codon and produces a truncated form of apolipoprotein. The C to A conversion occurs by
cytidine deaminase enzyme. This modification occurs only in certain cell types.
For example the editing of apolipo mRNA occurs in intestinal cells and does not happen in liver
cells. In some mRNAs, for example, the mRNA that encodes Cox II protein in trypanosomes,
four Us are inserted in a specific region that alters the reading frame of the message and the type
of protein produced by the mRNA. The editing in this case occurs by a special RNA called
guide RNA that consists of 40-80 nucleotides and with the help of other proteins like an
endonuclease and a ligase.
Fig. 6: Addition of Poly A tail to a eukaryotic mRNA
Ribosomes
Subunits, Composition and Morphology
Free ribosomes are found in the cytoplasm and are involved in the synthesis of cytosolic
proteins, whereas the membrane bound ribosomes are associated with the endoplasmic reticulum
and are involved in the synthesis of secretory proteins that are destined to reach various
organelles. In addition, ribosomes are found in the organelles like mitochondria and
choloroplasts. Organelle ribosomes resemble prokaryotic ribosomes. The subunits differ in their
size, length, width, composition, and in molecular mass. Protein synthesis occurs on the surface
of ribsomes. Ribosomes are RNA-protein complexes consisting of two subunits: a small (30S in
prokaryotes and 40S in eukaryotes) and large subunit (50S in prokaryotes or 60S n eukaryotes).
Together these subunits form monosomes (70S or 80S in prokaryotes and eukaryotes
respectively). When subjected to centrifugal force, the sedimentation value or Svedberg
coefficient (S) of the monosomes and their subunits are different. S Values are dependent not
only on mass but also the by shape and density. Although S values increase with molecular mass
of the ribosomal subunits, the S values of a monosome are not exactly add to its constituent
subunits. The S value of prokaryotic monosome is 70S and its constituent subunits are 30S and
50S. The small prokaryotic 30S subunit is divided into head, body and side bulge or platform
(Fig. 8). A distinct groove separates the head from the body. This subunit is composed of
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approximately 21 proteins (S1-S21, S refers to small subunit and they are named serially based
on their migration in gel electrophoresis), and 16S ribosomal RNA that play a critical role in the
recognition of start codon in prokaryotic mRNAs. The large 50S subunit is 160 kDa
approximately and it consists of 34 proteins (L1-L34) and two ribosomal RNAs: 23S (2900
bases) and 5S (120 bases) (Fig. 7). The large subunit is more isometric than the small subunit
with a linear size being equal to 200 to 230A0 in all directions. At the periphery, it has three
protuberances; the lateral is called the L7/L12 stalk, a central protuberance and a side lobe or L1
ridge (Fig. 8). Electron microscopic observations indicate that the appearance of eukaryotic and
prokaryotic ribosomal subunits is identical except that the 40S subunit has a protuberance
located on the head and appears to be bifurcated at the end of the body distal to the head.
Ribosome’s are not only involved in the actual peptide bond formation but a) provide the
necessary platform for the correct positioning of the tuna molecules carrying amino acids or
decollated tunas, b) facilitate the movement of tRNA on the ribosome and c) guide the accuracy
of the movement.
Processing of rRNA
Further, the size of the precursor of rRNA molecule is very long and is processed. The ribosomal
DNA of E. coli is transcribed as a 30S RNA precursor that is processed to yield 23S, 16S and 5S
RNA species. The 23S and 5S transcripts go into the composition of the 50S subunit, whereas
the 16S transcript is with the 30S small subunit (Fig. 7). The size of the precursor RNA transcript
in mammalian cells is 45S and is cleaved to give rise 28S, 18S and 5.8S RNA molecules. The
28S and 5.8S rRNA eventually become a part of the 60S subunit, and the 18S rRNA transcript
becomes part of the 40S subunit. The small ribosomal subunit of eukaryotes also contains 5S
rRNA but it is transcribed as a separate entity from a ribosomal RNA gene. The 5S rRNA is
present both in pro- and eukaryotic ribosomes and is conserved through evolution.
Figs. 7 and 8: Composition and morphology of eukaryotic and prokaryotic ribosomes
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Functions of ribosomal subunits
The small subunit plays a role in the initiation of protein synthesis process, and regulates the
fidelity of interaction between the three bases in the mRNA codon- tRNA anticodon. In contrast,
the large subunit is the center for the formation of peptide bonds between adjacent amino acids
and provides the pathway for the release of nascent or emerging proteins. Ribosomes consist of
three sites called aminoacyl (A), peptidyl (P) and the exit (E)- sites that interact with the amino
acylated tRNAs, peptidyl tRNA (tRNA carrying growing polypeptide), or with deacylated
tRNAs respectively. The distance between these sites is 20A0-50A0. Further, crystallographic
analyses of prokaryotic 30S ribosomal subunit reveal that 16S rRNA of 30S subunit modulates
the movement of mRNA-tRNA, and the ribosome dynamically changes its shape during different
functional states. These facts may explain why the structural features in tRNAs (see latter) are
conserved during evolution. Channels in the small subunit of ribosomes facilitate the entry and
exit of mRNAs and a channel in the large subunit facilitates the exit of nascent or newly made
polypeptide. Thus ribosome is not a static component. It is a dynamic molecular machine with
moving parts and a very complicated mechanism of action.
Ribosomal RNA, present at the interface between ribosomal subunits, not only plays a role in the
structure but also plays a role in two of the most important functions: a) decoding of mRNA and
b) addition of peptide bond between adjacent amino acids. Various studies suggest that the 16S
rRNA of the small ribosomal subunit scrutinizes the correctness of the codon-anticodon
interaction and is involved in decoding the mRNA sequence. The interaction between the first
two base pairs of codon-anticodon is checked more rigorously by the nucleotides in 16S rRNA
than the interaction between the third base pair. This fact relates to the wobble hypothesis (see
genetic code). 16S rRNA plays an important role in identifying the ‘start codon’ in mRNA in
prokaryotes (see latter). In contrast, the peptidyl transferase center in the 50S subunit that
catalyzes peptide bond formation between amino acids contains 23S rRNA. To perform these
functions (decoding and peptide bond formation), the crystal structure data of ribosomes suggest
that rRNA is located in the center or at the interface between large and small subunits of the
ribosome where as proteins are located mostly peripherally. Peptide bond formation does not
consume any additional energy or require ATP hydrolysis. However it uses the energy stored in
the acyl bond that was created during the charging of tRNA (see latter). Some of the ribosomal
proteins also shield the negatively charged RNA molecules.
Polysomes
A given messenger RNA may be translated by one or more than one ribosome. Accordingly the
number of copies of proteins produced will vary. A messenger RNA that is translated efficiently
by several ribosomes can give rise to a polyribosome complex. The total ribosomes in a cell are
distributed as small and large subunits (30s and 50S or 40S and 60S), monosomes (70S or 80S
i.e., single ribosome bound to mRNA), and polysomes (an mRNA bound by two to several
ribosomes). Isolation and analyses of the relative proportion of small subunits, large subunits,
monosomes and polyribosomes of total ribosomes reveal to some extent the protein synthetic
activity of the cell. Ribosomes isolated from a cell carrying active protein synthesis will have
very few subunits and monosomes but will have more polysomes. This is due to an increased
rate in the initiation of protein synthesis. Sometimes cells that are defective in protein synthesis
particularly at the level of elongation may also show up an increased proportion of polysomes
relative to their monosomes. Here the ribosomes pile up on mRNA and are released at a slow
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rate. Under such conditions, the polysome analyses can be correlated with protein synthesis in
vivo. One can estimate protein synthesis in cultured cells (control and treated cells) by
monitoring the incorporation of labeled amino acid like [S-35]- methionine or [C14] –leucine into
the acid precipitable protein based on the total uptake of the radioactive amino acid. In contrast, a
defect in the initiation step of protein synthesis in cells leads to the formation of a high
proportion of monosomes relative to polysomes.
Transfer RNA (tRNA)
Transfer RNAs are small RNAs compared to mRNAs and ribosomal RNAs. The tRNAs act like
adaptor molecules recognizing an amino acid on one side and the corresponding sequence
information in the mRNA sequence on the other side. Cells thus contain a minimum of twenty
tRNAs: one specifying each amino acid. However analyses of tRNAs and the genetic code (see
latter) have shown that cells generally contain more than twenty tRNAs and sometimes more
than one tRNA specifying an amino acid. Transfer RNAs contain 74-95 nucleotides. However
the precursor or the primary transcripts of tRNAs consist of around 125 nucleotides that are
processed to yield mature tRNAs. In both prokaryotes and eukryotes, the primary transcripts are
longer and contains introns that are processed out to give rise a functional tRNA. The various
regions of a typical mature tRNA are shown in Fig. 9.
Fig. 9: Transfer RNA with 5′ Phosphate and 3′ -OH group
Processing
It includes cleavage of the primary transcripts, splicing (joining of exons and removal of introns)
and addition of certain bases and modifications of certain bases. The processing is accomplished
by enzymes like RNAses P, D and E. As and when the primary transcript is made, it folds itself
into the stem-loop like structure. This folding is required and plays a critical role for the
nucleases to act upon the precursor molecule to remove certain nucleotides in the 5’ and 3’ends
of tRNAs. The processing occurs in an orderly manner and is somewhat different in prokaryotes
and eukaryotes. The pre-tRNA molecule in eukaryotes contains a 5’ leader sequence of about
15-16 nucleotides, an intron of 14-15 nucleotides and two additional nucleotides at the 3’end.
Removal of these nucleotides yields mature tRNA. The two most important features between the
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primary and processed transcripts between prokaryotic and eukaryotic tRNAs are a) the absence
of intron sequence near the anticodon, and b) the presence of CCA at the 3’end of pre-tRNA in
prokaryotes. In eukaryotes, the CCA nucleotides to which the cognate amino acid is attached are
added at the 3’end of mature tRNA by the action of nucleotidyl transferase enzyme.
Modified bases
Both pro- and eukaryotic tRNAs contain unusually rare and modified bases such as
ribothymidine, dihydrouridine, pseudouridine, 4-thiouridine, I-inosine, 1-methyl guanosine and
N6- isopentenyl adenosine (Fig. 10). The modified bases arise due to the activity of special
tRNA-modifying enzymes. Thymidine is generally present in DNA but it is also found in tRNA.
Uracil is modified to ribothymidine or pesudouridine by the addition of a methyl group or an
amino group respectively. Modifications of tRNA are required to affect the speed and accuracy
of protein synthesis. The modifications also play a critical role in maintaining proper reading
frame and for the movement of tRNAs from A-site to P-site (see latter). For example lysyl tRNA
(tRNALys with an anticodon UUU) recognizes AAA codon in mRNA and undergoes a
modification of N6-threonylcarbamoyladenosine at position 37 adjacent and 3’ to the anticodon
to bind AAA in the A-site of ribosomal 30S subunit.
Figs 10: Modified nucleotides in tRNAs
Fig. 11: 3-D structure of tRNA
The structures, but not the exact composition of the nucleotide sequences of tRNAs, are similar.
The secondary structure or the two-dimensional structure of tRNAs resembles to clover leaf (Fig.
9). Many of the nucleotides are complimentary to each other and form intermolecular hydrogen
bonds. As a result the tRNAs assume a typical structure which is critical for their function. The
tertiary or 3-D structure of tRNA resembles to L-shaped structure and is a result of nine
hydrogen bonds involving base pairing between several invariant residues (Fig. 11). The
interactions among bases in the T- and D- arms of tRNA facilitate the folding of the molecule
into an L-shaped structure with the anticodon at one end, and the amino acid acceptor at the other
end.
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Charging of tRNAs or Aminoacylation
The joining of an appropriate amino acid to the CCA containing 3’ end of amino acid acceptor
stem of the tRNA represents aminoacylation or charging of tRNAs. The reaction is catalyzed by
an aminoacyl synthetase and requires 2 high-energy bonds of ATP. The amino acylation of
tRNA is very specific and occurs in two steps. In the first step, the -COOH or carboxyl group of
an amino acid reacts with ATP producing aminoacyl AMP (adenylated amino acid, see Fig. 12)
and inorganic pyrophosphate (PPi). In step 2, the activated adenylated amino acid is transferred
to the 2’ or 3’ OH group of a sugar linked to an adenine base present at the 3’ end of an
appropriate tRNA. In the final reaction, the carboxyl group of the amino acid is linked to the 2’
or 3’ –OH sugar to a base present at the 3’ end of a tRNA. This base is always an adenine
(Fig.12).
Fig. 12: Aminoacylation of tRNA
Each aminoacyl synthetase recognizes specific amino acid and a tRNA. Cells have twenty
different synthetases, one for each of the 20 amino acids. The number of tRNAs present in a cell
may vary from 30-50 and thus exceed the number of amino acids. This suggests that there may
be more than one tRNA for some of the amino acids. These are called isoaccepting tRNAs with
different anticodons that accept the same amino acid (see below on wobble). The enzyme
aminoacyl synthetase recognizes specific sequences in tRNA particularly present in the D-loop,
anticodon loop and in the acceptor stem. For example, if the G3: U70 nucleotides of tRNAala are
used to replace the 3:70 base pair of tRNAcys or tRNAphe, then these modified tRNAs are
recognized by alanyl tRNA synthestase and charged with alanine suggesting that G3:U70 base
pair is a critical identity element in tRNAala for its specific synthetase.
Genetic Code
It refers to the relation between the four-letter nucleotide sequence information in mRNA to the
amino acid sequence information in proteins. The cracking of the genetic code is a history now.
15
Several theoretical considerations and elegant experimental results supported that a group of
three nucleotides, called a codon, specifies an amino acid. An mRNA that contains all of its
information in 4 letter nucleotides will have then 64 codons (43 = 64) that specify the twenty
naturally occurring amino acids. In contrast, a single nucleotide or groups of two or four
nucleotides would provide a maximum of 4, 16 or 256 codons respectively. While the single and
double letter codons cannot represent all of the twenty amino acids, the codon containing four
nucleotides was also not supported experimentally and theoretically as it uses maximally of the
four letters to specify twenty amino acids compared to the triplet code. In fact, the very early
mutagenesis experiments provided evidence to support the triplet code. It was shown that
bacteriophage T4 was unable to tolerate genetic changes that probably have altered one or two
bases. However, the phage was able to tolerate an alteration (deletion or insertion) of three
bases. Based on such genetic experiments, it is suggested that the genetic code is a triplet code.
In a triplet code, a change in one or two bases (insertion or deletion) alters the reading frame of
the mRNA and all the subsequent amino acids that are incorporated into the protein to the right
side of deletion (-) or insertion (+) (that is on the –COOH side of protein). However insertion of
three nucleotides into an mRNA sequence would change the coding ability of the message only
of those triplets at, and between the insertion of bases but not the amino acid sequence
downstream of the three inserted bases (see latter frame-shift mutations).
Cell-free translational systems
The genetic code was in fact deciphered before the cellular messenger RNAs were purified.
However by then cell-free translational systems were shown to support protein synthesis of the
endogenous mRNAs in vitro. The proteins prepared by such cell- free extracts can be labeled by
supplementing one radioactively labelled amino acid along with the other nineteen unlabelled
amino acids. The protein synthesis of the extracts carried out by endogenous mRNAs, or by
exogenously supplied mRNAs can be monitored by the incorporation of radioactively labeled
amino acid into the acid precipitable protein. This is possible because the cell-free protein
synthesizing systems contain all the machinery viz., the ribosomes, transfer RNAs, aminoacyl
synthetases that catalyze the joining of amino acids to tRNAs and other soluble factors
(initiation, elongation and termination factors) required for the protein synthesis. Additionally
the extracts are supplemented with ATP, GTP, and energy generating enzymes along with salts
like K+ and Mg2+. Such cell-free translational systems derived from bacterial extracts eventually
provided a means to determine the amino acid sequence of proteins coded by artificial and
natural templates. Subsequently these cell-free translational systems have been used as a source
for the preparation of reconstituted lysates to identify defective components of translational
machinery, for the purification of protein factors and enzymes, to study the mechanisms of
antibiotic actions, and to determine the mechanics and regulation of cytosolic and secretory
protein synthesis. Since many proteins are processed in physiological conditions and is difficult
to know the sequence if any in prepro-proteins (unprocessed proteins), translation of such
mRNAs in vitro systems devoid of the peptidases or proteases has provided a means to
determine the sequence of full length proteins. In addition, these translational systems are found
useful to evaluate the effects of antibiotics, toxins, and a host of other novel compounds for their
ability to affect protein synthesis at specific steps.
While bacterial systems and rat liver cell-free translational systems are used initially,
subsequently the preparation of heme-deficient rabbit reticulocyte lystates and cell-free
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translational systems derived from wheat embryos have unraveled some of the crucial regulatory
mechanisms involved in the initiation and elongation of mammalian cytosolic and secretory
protein synthesis. Wheat germ lysates, that do not have any endogenous mRNAs, were used to
determine the full length nature of a secretary protein, the role of a signal peptidase that
processes the signal sequence of a secretory protein, and also the role of other regulatory proteins
like signal recognition particle (SRP) that inhibits secretory protein synthesis, and docking
protein that relives the inhibition mediated by SRP. Similarly heme-deficient rabbit reticulocyte
lysates that carry mainly translational apparatus and mostly endogenous globin mRNA were used
to determine the importance of heme in the regulation of globin protein synthesis and to
elucidate the mechanism of inhibition of general translation in reticulocyte lysates in hemedeficient lysates. Micrococcal nuclease-treated reticulocyte lysates can also be used to determine
the translational products and activity of exogenously supplemented mRNAs. Micrococcal
nuclease is activated in the presence of calcium and can degrade the endogenous mRNA. Before
supplementing the exogenous mRNA, these nuclease treated lysates have to be treated with
EGTA that chelates calcium and inactivates the enzyme. While many animal systems can yield
cell –free translational systems with varying efficiencies, plant cell-free translational systems are
difficult to obtain because of the presence of vacuoles that contain hydrolytic enzymes that can
damage RNA.
Synthetic mRNA templates
The preparation of artificial template mRNAs is yet another important milestone in
understanding the genetic code. This has become possible because of the discovery of
polynucleotide phosphorylase enzyme. This enzyme is found to catalyze in fact the degradation
of RNAs to the corresponding ribonucleotides and inorganic phosphate in vivo. But,
interestingly, the purified enzyme has been observed to catalyze the formation of ribonucleoside
triphosphates (rNTPs) in the presence of high concentration of ribonucleoside diphosphates in
vitro. The incorporation of a ribonucleotide (A, U, G or C) is however dependent on the relative
amounts of the nucleotides used in the reaction mixture. For example, the chances of the
incorporation of nucleotide A (adenosine) only once in the template molecule in a reaction
mixture consisting of A and C in the ratio of 1: 4 are 1/5 whereas the chances for the
incorporation of C are 4/5, the highest. In other words, a template molecule with AAA will be
synthesized least and CCC will be the most abundant. Templates with two C s and one A (CCA,
ACC and CAC) will be the next abundant compared to two As and one C (AAC, CAA and
ACA). The codon sequence of these templates containing a mixture of nucleotides could
determine to some extent based on the relative abundance of the proteins produced by these
templates in cell-free translational systems, and then identify the amino acid sequences of the
protein (see below). However this method alone cannot give a precise sequence of information of
codons in mRNAs until additional information is available from other methods.
Amino acid analyses of polypeptides produced by synthetic templates
Synthetic mRNAs with single nucleotide or mRNAs (such as AAA…, CCC…, UUU…, and
GGG….), with alternating nucleotides (ACACACACACAAC), and with repeating sequences
(AACAACAACAACAACAAC) yielded a good amount of information to relate the nucleotide
sequence to the amino acid sequence in proteins. For example, translation of synthetic templates
containing single nucleotides (homopolymers) such as A, C, U or G in cell-free translational
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systems yielded polypeptides that contained a single amino acid sequence like lysine in the case
of polyA, phenylalanine in the case of poly U and proline in the case of poly C. Translation of
poly G is however found to be weak and produces polyglycine. The reason appears to be that the
poly G mRNAs lack proper folding. Translation of templates containing alternating nucleotides
like ACACACACAAC, yielded a polypeptide sequence that contains threonine and histidine
amino acids alternatively. To determine whether the codon sequence ACA or CAC is the cause
for the incorporation of threonine or histidine into the sequence, templates with repeating
trinucleotides such as AACAACAACAACAAC have been made and translated in cell-free
translational systems. Such sequences produced three types of polypeptides containing only
asparagine (Asn), threonine or glutamine. This is possible if the start site in the mRNA in each of
these cases is different. Now we know most of the natural mRNAs unlike synthetic mRNAs have
a ‘start’ site and ‘stop’ site. It is likely that if the synthetic mRNA sequence in the above case is
read as AAC AAC AAC AAC AAC, it may give a polypeptide that contains only Asn. But if the
first A is skipped, then the message is read as ACA ACA ACA ACA AC, and if the first two As
are skipped then the message will have a different sequence like CAA CAA CAA CAA C. The
common amino acid that is incorporated when the mRNAs containing AC repeats and AAC
repeats is threonine. Based on these results it is suggested that probably the ACA codon is
responsible for the incorporation of the amino acid threonine. However these results required
further validation. The reading frame in natural mRNAs is set by start codon which is usually
AUG and the code is generally nonoverlapping or in other words each nucleotide in an mRNA
belongs to a single reading frame.
Codon, charged tRNA and ribosome complexes can be captured on a filter
Researchers who were in the race to crack the genetic code had yet another interesting
observation wherein they found that a small triplet codon (like AUG, AAA or CUG etc.,) can
attract a complementary anticodon of a tRNA carrying the amino acid. The codon and anticodon
together can bind to ribosomes. Such codon-charged tRNA-ribosome complexes can be captured
on a filter paper. The complex can be identified because the amino acid bound to tRNA in the
complex is radioactive. This technique is found to be relatively more informative in deciphering
the genetic code because it is easy to prepare and handle small triplet nucleotide sequences than
long RNA templates. The synthetic triplet codons were incubated with a mixture of tRNAs each
carrying a radioactive amino acid. Small molecules like the tRNAs carrying amino acids whose
anticodons are not complementary to the codons cannot bind to the codon in the reaction and will
pass through the filter. This technique really facilitated to identify the recognition of 61 triplet
codons that are now known to recognize the twenty naturally occurring amino acids.
Wobble and degeneracy
Out of the total 64 codons, 61 codons specify 20 amino acids, and three codons UAA, UAG and
UGA specify stop codons. Since 61 codons specify the 20 naturally occurring amino acids, it
suggests that there are many amino acids that are specified by more than one codon. Hence it is
suggested that the genetic code is degenerate. Based on the number of sense codons, it is
suggested that there should be 61 tRNAs recognizing the 61 sense codons. However purification
and characterization of tRNAs revealed that many purified tRNAs are also found to recognize a)
more than one amino acid, and b) tRNAs contain some modified bases like inosine (derived from
adenine) in the anticodon that is different from the regular four bases present in RNAs. Transfer
RNAs with different anticodons that accept the same acid are called isosccepting tRNAs. Based
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on these observations, it is suggested that the first base in the 5’-anticodon of tRNA does not
follow strict Watson-Crick base-pair rule position while pairing with the 3’ end of codon. This
imperfect or nonstandard base pairing called wobble occurs between the third position of the
codon in mRNA and the first position of the anticodon in the tRNA (Fig. 13). Indeed analyses of
all the codons through the filter binding assays did show that 18 of the twenty amino acids have
more than one codon. Two of the amino acids, viz., methionine and tryptophan are specified by
one codon each. Among the others, three amino acids (serine, leucine and arginine) have 6
codons each accounting 18 of the 61 codons. Five of the amino acids (glycine, proline, alanine,
valine and threonine) have 4 codons each, and nine other amino acids (phenylalanine, tyrosine,
cysteine, histidine, glutamine, glutamic acid, asparagines, asparatic acid and lysine) are specified
by two codons. Isoleucine has three codons, and three codons UAA, UAG and UGA specify stop
codons thus accounting the 64 codons ( 2 + 18 + 20 + 18 + 3 + 3 = 64) in the genetic code (Fig.
14).
Fig. 13: Wobble
Indeed, analyses of many of the codons that specify the same amino acid have shown that these
codons differ in their third position. For example the codons specifying glycine are GGG, GGC,
GGA and GGU. In these cases, it is the last nucleotide residue or the nucleotide residue in the
third position of the codon is different. The degeneracy of the code or wobble may be
economical because it reduces the number of tRNAs from 61 to a minimum of 30 that are
required to recognize all the 61 triplet codons. The estimated number of tRNA species to be
present in bacteria is 30-40 and the number may be somewhat higher in some eukaryotes.
The code is mostly universal with the exception of few of the mitochondrial and protozoan
codons that behave slightly differently. For example mammalian mitochondrial UGA codes for
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trp instead of a stop signal. AUG codon is used in the cytoplasm to specify methionine during
initiation and elongation steps of protein synthesis. However AUA that specifies isoleucine in the
cytoplasm is used to specify internal methionine or methionine during the elongation step in
mitochondria. AGA and AGG codons that specify Arg amino acid in the cytoplasmic code
however specifies a stop signal in mitochondria. In fruit fly, AGA and AGG specify a serine
codon but not a stop codon or arginine. Again an analysis of the changes in coding specificity of
different codons reveals that a change in the base occurs primarily at the third position of the
codon in mRNA. Such changes may facilitate to decrease the number of tRNAs.
Fig. 14: Genetic Code
Mutations
Changes in the nucleotide sequences alter the genetic code. Point mutations arise because of
changes in a single nucleotide which are different from other kinds of drastic changes that occur
in DNA due to extensive insertions and deletions.
Missense mutation / Substitution mutation
A change in one codon (trinucleotide sequence) that specifies a particular amino acid to another
codon that specifies a different amino acid can occur by a single base change. The best example
is sickle cell anemia, a human genetic disease. Here the glutamic acid at position six in the
human β-globin is replaced by valine. The change is the result of a base change at the second
position of the codon GAA that codes for glutamic acid to GUA that codes for valine.
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Nonsense mutation
A change in the base of a codon specifying an amino acid to another base that results in a stop
codon. If it happens in the middle of a genetic message, it results in premature termination and
incomplete polypeptide synthesis.
Silent mutation
Often a change in the base that is present in the third position of a codon of an mRNA to another
base can result in a synonym that does not alter the amino acid sequence of the encoded protein.
Such changes are called silent mutations.
Neutral Mutation
It is a kind of missense mutation where the change in a base leads to a change in the
incorporation of a different amino acid that is chemically similar to the original one, and does
not influence the protein function.
Frame shift mutations
Insertion or deletion of one or more base pairs in the gene sequence affects the reading frame of
mRNA. As a consequence the amino acid sequence of the protein from the point of mutation to
the C-terminus will be modified. For example 5’…. ABC ABC ABC ABC ABC ABC ……3’
sequence in mRNA is modified by the insertion of a D for example can give a rise a message to
ABC ABC DAB CAB CAB CAB C. Even deletion of a base also alters the coding capacity not
only at the site of modification but also the rest of the message from the site of modification.
However in the above sequence if insertion or deletion of three bases (like D, E and F) instead of
one or two bases occurs then the coding specificity of the message is altered at and between the
insertions of these bases but the downstream sequences are not altered as shown below.
5’…. ABC ABC ABC ABC ABC ABC ……3’ is changed to 5’…. ABC DAB CAE BCA FBC
ABC ABC ……3’.
Suppressor Mutations
A suppressor mutation as the name indicates, suppresses the influence of another mutation. Thus
the organism that contains a suppressor mutation is a double mutant and it has two mutations:
one the original mutation and the second one being the suppressor mutation that will produce a
phenotype which is similar to the wild type. However this is not called reverse mutation. In
reverse mutation, whatever change occurred in the original nucleotide is restored. A suppressor
mutation occurs away from the original mutation site. The suppressor mutation can occur within
the same gene that contains already a mutation (intragenic suppressor) or in another gene
(intergenic suppressor). Both kinds of suppressor mutations block the effects of an earlier
mutation.
Gene density and overlapping genes
While the genome size and the approximate number of total genes present in an organism is
related to the complexity of an organism, gene density that refers to the average number of genes
per million bases (Mb) of genomic DNA is however high in lower organisms. An analysis of
gene density in different organisms reveals that highest gene densities are found in viruses where
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sometimes both strands of DNA are used and encode overlapping genes. In bacteria, the gene
density is very high compared to eukaryotes. Overlapping genes are found in many viruses
where the genome size is small. Such overlapping genes produce more than one protein
depending on the reading frame of the gene and the start site. In prokaryotes, the mRNAs are
polycistronic, and in such cases the ribosomes have to be able to find the start codon before they
are detached. In eukaryotes, alternative RNA splicing is used to generate variant proteins from
one gene.
Protein Synthesis
The machinery of protein synthesis consists of ribosomes, subunits, mRNAs, tRNAs and amino
acyl synthetases, protein factors, amino acids, ATP and GTP. For convenience, the process is
divided into four steps: initiation, elongation, termination and ribosome recycling. Although, the
overall process of protein synthesis is similar, several differences exist among prokaryotes,
archea and in eukaryotes. The description here mostly refers to the general protein synthesis and
to some important differences between prokaryotes and eukaryotes.
Initiation
Initiator and elongator methionyl tRNAs and formylation
Initiation of protein synthesis in vivo is a very precise process. In spite of several nucleotides
present in the 5’end of mRNA, ribosome skips these codons and initiates the synthesis from the
first AUG codon that it encounters on the 5’ side. Since AUG codes for methionine, it is the first
amino acid to be incorporated in any protein. Rarely GUG may be used that codes for valine.
However two tRNAs are involved in bringing the amino acid methionine: one is initiator
methionyl tRNA (fMet-tRNAfMet in prokaryotes) and another one is elongator methionyl
tRNA(Met-tRNAmMet).The difference is that the methionine bound to the initiator tRNA in
prokaryotes is formylated by a transformylase enzyme and the formyl group comes from N10
tetrahydrofolate. The methionine carried by the elongator tRNA is not formylated. Athough two
tRNAs exist in eukaryotes for methionine as in prokaryotes, the methionine bound to initiator is
not formylated in eukaryotes. The sequence differences between the three tRNAs are illustrated
in Fig. 15. Further, IF2 initiation factor in prokaryotes and eIF2 in eukaryotes facilitate the
joining of initiator tRNA carrying methionine to the respective small ribosomal subunits in the
presence of GTP. The elongator methionyl tRNA (Met-tRNAmMet) and other amino acylated
tRNAs are delivered to the 70S initiation complexes by elongation factor EF.Tu in prokaryotes
and by EF1 in eukaryotes.
Formation of 70S initiation complex
The process of initiation occurs in an orderly manner with the help of initiation factors (IFs) (Fig.
16). While the prokaryotic initiation factors are called IFs the eukaryotic factors are represented
as eIFs. The final product of initiation step is the formation of a 70S in prokaryotes and 80S
ribosome complex in eukaryotes with the corresponding initiator tRNA properly positioned on
the first AUG codon of mRNA at the P site of ribosome. Initiation in prokaryotes starts with the
small ribosomal subunit (30S) that is bound by IF1, IF2 and IF3 factors. The IFs interact with
domains in small ribosomal subunit that will subsequently interact with the amino acylated,
peptidyl and deacylated tRNAs respectively i.e., A site, P site or E site in ribosome. While IF1
binds to the A site in the ribosomal subunit that will eventually harbor an incoming amino
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acylated tRNA, IF2 interacts with the A and P sites, and IF3 interacts with E site of the
ribosome. To begin with, the f-Met-tRNAfMet joins IF2.GTP associated with the 30S subunit in a
codon-independent manner. The 30S complex also binds mRNA through its ribosome binding
site (RBS). Although in eukaryotes, the Met-tRNAiMet joins the 40S ribosome before the mRNA
joins, the order of these two events in prokaryotes is not clear. RBS is also called as ShineDalgarno sequence in prokaryotes. It is a 7-8 nucleotides long sequence in mRNA located
upstream of the initiation codon (AUG). It contains a polypurine rich sequence (Ex:
5………..AGGGGAAAU--- AUG……..3’ in E. coli trpA gene ) that interacts with the
polypyrimidine sequence (3’ ….CCUCCU…..5’) present in the 16S ribosomal RNA of the 30S
subunit through complementary base pairing. This will facilitate 30S subunit of ribosome to
attach the mRNA and to position itself over the initiation codon directly.
Fig. 15: Important sequence differences in different Methionyl tRNAs
However this unstable 30S complex (30S ribosome.IF1.IF3.IF2.GTP.fMet-tRNAfMet. mRNA)
undergoes a stable conformation that promotes codon-anticodon interaction. This triggers a
conformational change in 30S ribosome and facilitates the release of IF3 (an anti-association
factor) so that the 50S subunit joins the 30S initiation complex to form 70S initiation complex. A
GTPase facilitates the hydrolysis of GTP bound to IF2 to GDP and that will facilitate the release
of both IF2 and IF1 from the 30S subunit. At the end of initiation, 70S ribosome complex devoid
of initiation factors is formed with fMet-tRNAfMet positioned in the P-site of ribosome at the start
codon (Fig. 16).
Functions of Initiation Factors
All three initiation factors help to position the initiator tRNA in the ‘P’ site of ribosome on the
start AUG codon in mRNA. IF3 especially stabilizes the 30S initiation complex. IF1, the
smallest protein factor among all the three initiation factors (8.2 kDa in E. Coli) promotes the
interaction between IF2 and 30S subunit and more specifically the interaction of IF2. fMet.
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tRNA.GTP with the initiation codon of the mRNA in the P-site. IF1 through its interaction with
the A-site blocks the association of amino acylated tRNAs to the A-site until the formation of
70S initiation complex. IF2 is the largest initiation factor (approx 89-90 kDa) with highest
affinity to ribosomes compared to the two other factors and joins specifically fMet-tRNAfMet.
The complex IF2.GTP will promote the association of 30S initiation complex with 50S subunit
to form 70S initiation complex. Hydrolysis of GTP bound to IF2 by a GTPase occurs in the
presence of ribosomes. It is not clear whether the GTPase activity is intrinsic to IF2 protein and
is triggered upon the association of 30S pre-initiation complex with the 50S subunit. The GTP
hydrolysis however at the end of initiation facilitates a) the release of IF2.GDP from the 30S
initiation complex, and b) the adjustment of initiator tRNA in the P-site of ribosome with proper
codon-anticodon pairing. GTP hydrolysis by a GTPase however occurs only after the subunits
are joined. Hydrolysis of the GTP may cause a conformational change in ribosome that would
facilitate the release of all the factors. IF2.GDP that is produced at this stage has reduced affinity
for ribosome and is also released along with IF1. IF2 like factor is also recently discovered in
eukaryotes and is called eIF5B with a GTPase activity. It plays a role in the joining of large
subunit with the small subunit at the end of initiation in eukaryotes (see latter). IF3 is a 20.4 kDa
protein. IF3 bound 30S subunit cannot join the 50S subunit to form 70S initiation complex
unless the factor is released. Thus, it prevents association of the ribosome subunits. It also
promotes the dissociation of noninitiator aminoacylated tRNAs binding to the P-site of 30S
ribosomal subunit, the dissociation of 70S complexes, and in the recycling of ribosomal subunits
at the end of synthesis.
Fig. 16: Initiation of Protein Synthesis in Prokaryotes
Elongation
This occurs in three steps:
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(i)
(ii)
(iii)
delivery of the amino acylated tRNA to the A site of 70S ribosome complex;
peptide bond formation between adjacent amino acids by petidyl transferase (ribosomal
RNA or ribozyme); and
the movement of mRNA by three nucleotides.
i. EF.Tu/Ts factor promotes the joining of amino acylated tRNA to the A-site in ribosome
In step1, an aminoacylated tRNA attachment to the A site in 70S ribosome is catalyzed by
elongation factor EF.Tu and GTP. Unlike IF2, EF.Tu has a high affinity for GDP. However GDP
inhibits the joining of aminoacylated tRNA to EF.Tu. Hence exchange of GTP for the bound
GDP is critical. This GDP/GTP exchange is catalyzed by a guanine nucleotide exchange factor
called EF.Ts. EF.Tu binds the 3’end of tRNAs and protects the attached amino acid from
entering into the peptide bond formation. After the delivery of aminoacylated tRNA into the A
site of ribosome, the GTP bound to EF.Tu is hydrolyzed and the EF.Tu.GDP is released. The
latter is recycled by EF.Ts and GTP to form EF.Tu.GTP that is competent to join aminoacylated
tRNA. The GTPase activity that hydrolyzes the GTP bound to EF.Tu is stimulated when the
ternary complex, EF.Tu.GTP. aa tRNA, joins the ‘A’ site of the ribosome and interacts with the
factor binding center of the ribosome. Further the efficiency of GTPase activity is high and
dependent on the correct base pairing between codon-anticodon. Thus it acts as one of the
mechanisms to ensure proper codon-anticodon interactions.
ii.Ribosomal RNA of the large subunit promotes the Peptide bond formation
The next step in elongation is the formation of peptide bond between adjacent amino acids. It
occurs at the peptidyl transferase center present in the large subunit. The peptide bond formation
takes place between the amino acid present at the 3’ end of tRNA of the growing polypeptide
chain in the ‘P’ site and the aminoacylated tRNA present in the ‘A’ site. In this process, the
growing polypeptide attached to the peptidyl tRNA is transferred to the tRNA present in the Asite (contains a newly arrived amino acylated tRNA). The peptide bond formation requires Nterminus of the protein to be synthesized before the C-terminus. After the addition of peptide
bond by the peptidyl transferase center of the large ribosome subunit, the peptidyl tRNA is
deacylated. For a long time it is believed that one of the proteins of the large subunit is involved
in the catalysis of peptide bond formation. However the current evidence suggests the large size
ribosomal RNA (23S in prokaryotes and 28S in eukaryotes) of the large ribosomal subunit
catalyzes the peptide bond formation. Although the exact mechanism is yet to be determined, it
is suggested that the nitrogen of the nucleotides that accepts a hydrogen atom from the α-amino
group of the amino acid bound to amino acylated tRNA can act as a strong nucleophile and can
attack the carbonyl group of the growing polypeptide attached to the peptidyl tRNA (as shown
below). Since no fresh energy input is required to promote peptide bond formation, the reaction
is driven by high energy acyl bond that is formed during the tRNA charging (Fig.17).
iii. Translocation of tRNA and mRNA are promoted by EF-G
After the formation of peptide bond, the tRNA in the ‘P’site is deacylated and the growing
polypeptide chain joins the ‘A’ site. Then the mRNA moves by three nucleotides to bring in the
next codon. The deacylated tRNA moves to the ‘E’ site whereas the growing polypeptide chain
located at this point in ‘A’ site moves to the ‘P’ site thus allowing room for a new amino
acylated tRNA to enter the ‘A’ site. The evacuation of deacylated tRNA from the E-site and the
25
movement of mRNA by one codon relative to the ribosome requires the mediation of elongation
factor-G (EF-G) and GTP. EF-G thus acts like a translocase enzyme and drives the translocation
of tRNA and mRNA. The translocation step requires the hydrolysis GTP bound to EF-G. The
hydrolysis of GTP is stimulated when EF-G contacts the factor-binding center of the ribosome.
GTP hydrolysis triggers a conformational change in the ribosome that facilitates the translocation
of the growing polypeptide chain from A site to P-site and also formation of EF-G.GDP. In fact,
the latter has reduced affinity to ribosome compared to EF-G.GTP complex. EF.G.GDP thus
formed replaces the tRNA in the A-site to P-site and occupies the A-site. This will also facilitate
the movement of deacylated tRNA from P-site to E-site. This movement of tRNA in the A-site
mediates the movement of mRNA by one codon or three bases This movement of mRNA during
translocation has been further supported by the fact that movement of rare frame shift tRNAs
that have four nucleotides in their anticodon region can move the mRNA by four base pairs.
Thus the translocation step is promoted by EF-G.GDP complex that binds to the A-site.
Interestingly, EF.G.GDP structure appears to resemble EF.Tu.GTP.aa.tRNA complex which also
binds to the A-site. This is a kind of molecular mimicry (Fig. 18).
Fig. 17: Peptide Bond Formation
iv. Energy requirements in the elongation cycle
The elongation cycle consisting of joining of charged aminoacylated tRNA to the A-site, peptide
bond formation and translocation of tRNA and mRNA requires the consumption four high
energy bonds in each round of elongation cycle or for the incorporation of one amino acid. Two
of these high energy bonds come from one molecule of ATP and are used in the charging of
amino acid to tRNA, and two of them come from two molecules of GTP. The high-energy bonds
of GTP are used in the joining of amino acylated tRNA to the A-site in ribosome, in the fidelity
of translation and in the translocation step. In contrast, a minimum of three high-energy bonds is
required in the initiation cycle of prokaryotes. Two of these bonds that come from ATP are used
in the charging of initiator tRNA and one molecule of GTP is hydrolyzed soon after proper
codon-anticodon interaction, before the release of the factors and the formation of 70S initiation
complex. In eukaryotes, however there appears to be additional energy requirement to unwind
the secondary structure in mRNA and in the subunit- joining step as mentioned above.
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Termination
Release factors (RFs)
Two classes of release factors (RFs) have been identified. Class1RFs identify stop codons and
facilitate the release of newly made polypeptide chains. When a ribosome reaches one of the
three stop codons in mRNA, no amino acylated tRNA enters its A-site. Instead termination
factors designated as release factors (RFs) recognize the three stop codons (Fig. 18). Prokaryotes
contain two class 1 factors: RF1 and RF2 that recognize three stop codons. RF1 recognizes
UAG stop codon, whereas UAA stop codon is recognized by both RF1 and RF2. The third stop
codon, UGA, is however recognized by RF2. Unlike in prokaryotes, eukaryotes contain only one
factor called eRF1 that recognizes all the three stop codons.
Fig. 18
Class1 release factors
The class1 release factors carry evidently two functions: one of them is the recognition of the
stop codon and the second function involves the release or hydrolysis of the nascent peptidyl
chain. A sequence of three amino acids (SPF i.e serine-proline and phenylalanine) in the RF
protein recognizes the stop codon and thus serves as a peptide anticodon. The other function
(release of the nascent polypeptide) evidently requires a conserved GGQ (glycine-glycineglutamine) sequence in RF. These two regions are close to each other in the absence of a
ribosome but in the presence of a ribosome, the release factor undergoes a conformational
27
change that would space these two regions appropriately to serve the two functions as mentioned
below.
Class II RFs
Under the category of class II, only one release factor is identified and is designated as RF3 in
prokaryotes and eRF3 in eukaryotes. Class II factors stimulate the dissociation of class I factors
from the ribosome and they require GTP.
Ribosome recycling
After the release of polypeptide chain and RFs, the ribosme is bound by mRNA and deacylated
tRNAs in the P and E-sites. The dissociation of ribosomes into subunits, the removal of
deacylated tRNAs and mRNAs is aided by ribosome releasing factor, RRF as has been
demonstrated in prokaryotes. It resembles to tRNA and thus it can join the A-site. Elongation
factor, EF.G, that mimics like a tRNA joins the RRF bound to ribosomes. The binding of these
factors somehow facilitates the evacuation of deacylated tRNAs, subunit dissociation and mRNA
release. Interestingly, initiation factor 3 (IF3) may also join the small subunit and prevents the
joining or association of the large subunit with the small subunit and aids in mRNA dissociation
(Fig. 18).
Differences in the initiation between eukaryotes and prokaryotes
The basic steps in the initiation of protein synthesis are the same in prokaryotes and eukaryotes,
i.e., to bring the initiator tRNA and mRNA onto the small ribosomal subunit, identification of
start codon, and the joining of large subunit with the preinitiation complex to form 70S or 80S
initiation complex. The components and the mechanisms however are more complicated in
eukaryotes than in bacteria. In eukaryotes, the methionine bound to the initiator tRNA is not
formylated and the steps joining the initiator tRNA and mRNA appear to be occurring one after
another.
In eukaryotes, there are a dozen protein factors involved in the initiation that are listed in table-1
Eukaryotic initiation factor 2 (eIF2) like its prokaryotic counter part IF2, facilitates the joining of
methionyl initiator tRNA to 40S subunits that is bound by a multisubunit eIF3 (an anti
association factor) and eIF1A and eIF1. The whole complex is now called the multifactor
complex. However, eIF2 has a very high affinity for GDP than for GTP in the presence of
physiological concentrations of Mg2+and GDP inhibits the joining of eIF2 to initiator tRNA
carrying methionine. Hence eukaryotic cells have a GDP/GTP exchange factor called eIF2B that
catalyzes the exchange of GTP for GDP bound to eIF2.
The multifactor complex then joins mRNA bound by eIF4F complex. This complex contains
three different proteins viz., eIF4G, eIF4E and eIF4A with different functions. While the
complex containing three proteins is required for the translation of eukaryotic mRNAs that
contain 5’cap, eIF4A with the energy coming from ATP hydrolysis acts as a helicase and
facilitates the unwinding of any secondary structure in mRNA. The mRNA is held in place by
eIF4E and eIF4G is a multivalent adaptor complex interacting with eIF4E, 4A, eIF3 of
multifactor complex and also with the polyA binding protein that binds the poly A tail of
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eukaryotic mRNAs. In prokaryotes, no ATP hydrolysis occurs to unwind the mRNA structure as
in prokaryotes. The first ATP hydrolysis occurs in all organisms is at the charging of initiator
tRNA.
In addition, the eukaryotic mRNAs do not have anti- Shine-Dalgarno sequence that allows easy
recognition of the start AUG on mRNA by 18S rRNA. Eukaryotic mRNAs have 5’ caps and 3’
poly A tails because of which the joining of 43S complex (40S ribosome-Met-tRNAiMet complex
bound by several factors) is more complicated than in prokaryotes. An analysis of the sequences
around the initiation sites in eukaryotic mRNAs (plants, yeast and mammals) indicates an A
preceding the start AUG and G after the start AUG (5’ …….A…AUGG……3’ sequence around
the initiating AUG in eukaryotic mRNA). In mammals, the recognition of AUG start codon
critically requires the surrounding sequence, GCC (A/G) CC AUG G. eIF1 and eIF1A are the
factors implicated in identifying the initiation codon selection and in maintaining the
translational fidelity.
Further, in eukaryotes, the proper positioning of the initiator tRNA anticodon on the start codon
of 48S initiation complex stimulates the GTPase activity of associated with one of the subunits
of eIF2 by a protein called eIF5. Hence the latter is called GTPase activating protein (GAP) that
hydrolyzes GTP bound to eIF2 to GDP and releases eIF2.GDP. However the 60S subunit joining
to 48S initiation complex in eukaryotes requires yet another GTP hydrolysis that is promoted by
eIF5B, a factor that has sequence homology with prokaryotic IF2.
In addition to 5’ m7GpppN (methylated guanosine cap) and 3’ poly A tail that promote
translation, many eukaryotic mRNAs carry secondary and tertiary structural elements like hair
pins and pseudoknots respectively, internal ribosome entry sequences (IRESs) at the 5’end that
facilitate cap-independent translation, small upstream open reading frames (uORFs) that act
normally as negative regulators of translation from the main ORF, and also special nucleotide
sequences both at the 5’ and 3’ ends of mRNA that can bind to RNA or proteins. These sequence
elements in mRNAs regulate translation.
In the elongation cycle, eukaryotes have eEF1A and eEF1B (α, β and γ) are comparable to their
function to prokaryotic EF-Tu and –Ts respectively whereas eEF-2 in eukaryotes is comparable
prokaryotic EF-G. Further the activity of many of the eukaryotic, but not prokaryotic, initiation
and elongation factors is regulated by phosphorylation-dephosphorylation.
Average rates of amino acid incorporation in a bacterium like E. coli will be 15-20 aa /sec which
is equivalent to a decoding of 15-20 codons or 45-60 nucleotides in an mRNA/sec. Apparently
the rate is far slower in eukaryotes, and is about 5-10aa /second at370C
Inhibitors
Antibiotics (produced by bacteria and yeast and their man made versions that are used to treat
various bacterial infections), toxins (proteins produced by pathogenic bacteria such as cholera,
diptheria etc.,), lectins (carbohydrate-binding proteins originally found in plants) and
physiological agents such as interferons (natural proteins produced in response to viral infection
in human cells) inhibit protein synthesis at the initiation, elongation or at termination level.
The ribosome is an important target for a wide variety of antibiotics. Broad spectrum antibiotics
such as streptomycin and tetracycline were of great clinical importance when they were first
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discovered. However with the increased resistant strains of bacteria, there is a real need to
understand details of how these antibiotics interact with the ribosome. Many of the antibiotics
primarily bind to ribosomal RNA and inhibit protein synthesis. Most of the clinically useful
antibiotics target the large subunit 50S in the peptidyl transferase center, its vicinity or at the
entrance to the ribosome tunnel. Some of the antibiotics bind to the 16S rRNA of the 30S subunit
and still interfere with the translocation step than initiation step as mentioned below.
Table 1: Translational initiation factors in eukaryotes and their functions
Eukaryotic
initiation factor
Function
eIF1
eIF1A
‘Start’ AUG codon recognition
Multiple roles? Facilitates Met-tRNAiMet
binding to 40S subunit
Facilitates the joining of Met-tRNAiMet to 40S
subunit, has higher affinity for GDP. Contains
GTPase activity, interacts with mRNA and the
other proteins.
GDP/GTP exchange factor. Functionally it is
like Ts factor in prokaryotic elongation cycle.
Exchanges GTP for GDP bound to eIF2.
Recycles in active eIF2.GDP to active
eIF2.GTP so that eIF2 joins initiator tRNA.
Promotes Met-tRNAiMet and mRNA binding to
40S.
Helicase, unwinds mRNA secondary structure
Promotes the helicase activity of eIF4A
Binds 5’ cap of the mRNA
eIF2
(three
subunit
protei:
α, β and γ)
eIF2B
(heteropentameric )
eIF3
(Multi
subunit protein)
eIF4A
eIF4B
eIF4E
eIF4F
eIF4G
eIF5
eIF5B
PABP
Complex of cap binding proteins: eIF4A, 4B,
4E and 4G
Scaffolding or multivalent adaptor protein that
interacts with proteins bound to 5’ and 3’ end
of mRNAs.
Involved in AUG recognition and activates the
GTPase activity of eIF2.
In the joining of 60S to 48S complex to form
80S initiation complex. Its sequence resembles
to IF2 that is involved in Met-tRNAfMet
binding.
Poly A- binding protein that provides contact
for eIF4G so that mRNA can be circularised
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Streptomycin interacts with the phosphate backbone of 16S rRNA and S12 protein and
hypothesized to increase the binding of non cognate-tRNAs that would lead to misincorporation
of wrong amino acids into the polypeptide chains.
Tetracycline was found to be a multi-site antibiotic with inhibitory action. It interferes with the
A-site tRNA binding. Tetracycline binds to two functional sites in the 30S subunit. It has a major
binding site at the A site of tRNA, and a minor binding site near 16S rRNA. It is suggested that
the binding of tetracycline interferes in the binding of amino acylated tRNA at the A-site during
translation. In the presence of tetracycline, GTP hydrolysis of the ternary complex,
EF.Tu.GTP.aatRNA occurs in an unproductive manner and the amino acylated tRNA is ejected
from the A-site without peptide bond formation due to a stearic clash with the antibiotic.
Tetracycline thus prevents protein synthesis but facilitates GTP hydrolysis. The GTP hydrolysis
without protein synthesis is energetically very expensive to the cell. Consistent with the
inhibition of tRNA binding to the A-site during translation, tetracycline also inhibits the joining
of releasing factors RF-1 and RF-2 during termination regardless of the stop codon. It is
suggested that the binding of tetracycline on the second site i.e., at 16S rRNA on 30S subunit
increases binding of non- or near-cognate tRNAs that would lead to misincorporation of wrong
amino acids into the polypeptide chains as has been suggested for streptomycin.
Pactamycin binds to two conserved nucleotides G693 and C795 that are universally conserved in
the 16S rRNA of the small subunit and protects them from chemical modification and thereby
reduces the flexibility of 30S subunit by locking two of the 16S rRNA helices 23 and 24 together
and inhibits translocation but not initiation as predicted in earlier studies.
Edeine is found to induce base-pairing between G693 and C795 conserved residues of 16S rRNA
and inhibits tRNA binding to the P-site by preventing codon-anticodon interaction. Interestingly,
pactamycin can rebreak the base pair formed by edeine. These findings with these two antibiotics
antagonizing each other’s function highlights the importance of the G693:C795 base pair in 16S
rRNA for the tRNA-binding at the P-site of the ribosome and its importance in translocation.
Edeine is also found to act like streptomycin in promoting translational misreading. Both edeine
and pactamycin are universal inhibitors.
Puromycin that resembles the 3’ end of amino acylated tRNA can join the ribosomal A-site,
accepts the growing polypeptide chain and then the next step of elongation is blocked. The
growing polypeptide is released prematuredly. Puromycin affects both bacterial and eukaryotic
translation.
Kirromycin inhibits the conformational changes in ribosome that occur and facilitate the release
of EF.Tu from ribosome after the hydrolysis of GTP. If the EF-Tu factor is not released then
another EF-Tu carrying another aminoacylated tRNA cannot be delivered to the 70S complex.
Fusidic acid prevents the release of EF-G.GDP from prokaryotic ribosome so that the next step
in translation is arrested.
Cycloheximide an inhibitor of peptidyl transferase activity is associated with the 60S subunit of
eukaryotes. It prevents peptide bond formation. It blocks elongation and facilitates the formation
of polyribosome complex. The polysome formation is a result of decreased elongation rather
than increased initiation.
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Chloramphenicol a prokaryotic translational inhibitor, reduces productive positioning of the
aminoacylated tRNA at the A-site and thereby interferes in peptide bond formation.
Sparsomycin an universal inhibitor of translation, promotes stacking interactions in the rRNA
thereby promoting conformational alterations in the A- and P- sites of ribosome. Its primary
action relies on the interaction with the P-site tRNA and it enhances the affinity of tRNA for the
P-site (i.e., the tRNA movement is reduced from P to E-site) because of which translocation is
blocked and protein synthesis is inhibited.
Aminoglycosides (streptomycin, amikacin, tobramycin and neomycin) bind to the 30S subunit
and block initiation of protein synthesis by forming aberrant initiation complexes. In addition,
they cause miscoding at the aminoacyl-tRNA-mRNA step.
Macrolides (erythromycin, clarithromycin and roxithromycin) containing a 14 or 15 or 16membered lactone ring, to which one or more deoxy sugars, usually cladinose and desosamine,
are attached, bind to 23S ribosomal RNA at the peptidyl transferase center and act by producing
a stearic blockage of the ribosome exit tunnel and hamper the progression of nascent chains.
They generally bind to A2058, a ribosomal RNA nucleotide.
Toxins produced by Diptheria and Pseudomonos modify elongation factor 2 in eukaryotes
(eEF2) and inhibit translation. These toxins contain ADP-ribosyl transferase activity that
transfers the ADP-ribose from NAD (nicotinamide adenine dinucleotide) to a modified histidine
amino acid residue of the elongation factor 2 (EF2). ADP-ribosylated eEF2 the elongation step
in protein synthesis.
Ribosome inactivating proteins (RIPs) are plant proteins that inhibit of protein synthesis. Two
types of RIPs are found. Type1 RIPs are N-glycosylated monomeric non-cytotoxic proteins.
Type 2 RIPS are dimeric consisting of two different chains linked by a disulfide bridge. One of
them is an enzymatic chain and the other is a lectin that recognizes membrane sugars, mostly
galactose residues. Many type2 RIPs such as ricin, abrin, viscumin, modeccin and volkensin
have been isolated to date. Ricin for example produced by castor seeds is a most potent among
the toxins. Both types of RIPs have N-glycosidase activity and can act on the largest rRNA of the
ribosomes.
Interferons are cytokines with a particularly direct impact on protein synthesis in the virusinfected cells. They induce in turn enzymes like double-stranded RNA-dependent protein kinase
(PKR) and 2’ 5’ oligo adenylate synthetase (2-5A Synthetase) that act at the cellular level as first
line defenses against viral infection. Both these latent forms of enzymes are activated by doublestranded RNA through different mechanisms. In the presence of dsRNA, activated 2-5A
synthetase produces a series of short 2’-5’ linked oligonucleotides pppA(pA)n, where n is
generally 2 or 3. These short 2-5A oligonucleotides activate RNAse L, which degrades RNA
mainly by cutting at the 3’side. The other enzyme PKR is a serine-threonine kinase and is also
activated in the presence of dsRNA. Activated kinase phosphorylates the alpha subunit of
eukaryotic initiation factor 2 (eIF2α) and inhibits the initiation of protein synthesis of general
mRNAs. In fact phosphorylation of eIF2α is a stress signal and occurs in heme-deficiency, virusinfection, amino acid starvation and during accumulation of unfolded proteins through the
activation of specific eIF2α kinases such as heme-regulated kinase (HRI), PKR, PERK (PKR32
like ER-resident kinase) and GCN2 (general control non-derepressible) respectively. In fact both
HRI and PERK are activated in response to the accumulation of denatured proteins or unfolded
proteins. Many times when the synthesis exceeds the folding, proteins cannot fold properly.
Under such conditions, endoplasmic reticulum that is involved in the synthesis of secretory
proteins evokes a complex signaling pathway called an unfolded protein response (UPR) that is
primarily adaptive in nature. The UPR has two arms: translational attenuation that is mediated by
the phosphorylation of the α-subunit of eIF2 and a transcriptional induction of chaperones that
correct the protein folding.
Translational regulation
Translational control is defined as a change in the efficiency and utilization of mRNAs. The
synthesis of different proteins is altered during development, differentiation, virus infection,
hormonal treatment and depending on various external conditions. In many of these cases
mentioned above, in addition to changes in the RNA synthesis (transcription), the changes in
translational apparatus also regulate the synthesis of proteins. Very early experiments have
shown that eggs contain mRNA that is not translated until fertilization. The rate of protein
synthesis in these egg cells is shot up soon after fertilization. Until fertilization these mRNAs are
masked with proteins (mRNP complexes) that keep the mRNAs silent. Deproteinized egg
mRNA can be translated efficiently. In many animals, the first few hours of life after fertilization
proceed with very little transcriptional activity. Maternal mRNAs present before fertilization
play a crucial role in early decisions. Translational controls are important for establishing body
axes i.e the anterior-posterior and dorsal and ventral-axes of the mature organisms. Similarly
heme-deficiency has been shown to inhibit the translation of hemoglobin mRNA in reticulocytes,
or in their lysates (cell extracts). Addition of hemin, the iron protoporphyrin compound,
stimulates the hemoglobin synthesis in the cell-free translational systems derived from hemedeficient reticulocytes. Heat shock for example inhibits translation of normal mRNAs but
facilitates the translation of heat shock mRNAs. Virus infection limits the synthesis of many host
proteins but facilitate the synthesis of viral proteins. In most of these cases the regulation of
protein synthesis occurs at the initiation level. However, protein synthesis is also found regulated
at the elongation level. The best examples are the secretory proteins that are made on the surface
of endoplasmic reticulum. The secretory proteins interact with a ribonucleoprotein complex
called signal recognition particle (SRP) during the elongation phase. SRP is a ribonucleoprotein
complex consisting of 7S RNA. The interaction of growing secretory polypeptide with SRP
arrests the synthesis of the secretory polypeptide. The block in elongation is relieved in the
presence of ER membrane protein called docking protein. The latter removes SRP bound to the
growing polypeptide. This kind of a regulation appears to be essential for many of the digestive
enzymes (most of them are secretory proteins) whose synthesis occurs only after a meal. When
the enzyme is not needed the synthesis does not occur or it is arrested at the level of elongation.
An analysis of these examples provided a wealth of information to understand the molecular
mechanisms (role of different protein factors, small nuclear RNAs, covalent modifications of
proteins, critical elements in mRNA structure) regulating translation in development,
differentiation, health and disease.
Reasons for translational regulation
In spite of the fact that translation or protein synthesis is the last step in the overall gene
expression, cells have evolved regulatory mechanisms that control overall translation as
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mentioned above. These regulatory mechanisms affect the posttranscriptional modifications of
mRNAs, transport of mRNAs to cytosol, assembly of amino acids, protein folding and
degradation. It appears the cell can apply translational regulation much more easily because it
takes very little time than regulating the transcription. Translational regulation is quite often
more focused, fast and flexibile affecting the translation of selective messages or whole class of
mRNAs as in heat shock and virus-infected host cells. Along with transcriptional regulation,
translational controls offer to fine-tune the regulation of gene expression many times. In many
systems like egg cells, reticulocytes and RNA viruses where there is limited transcriptional
regulation, translational controls play most important role. This fact suggests that probably
translational controls are in existence prior to the evolution of transcriptional controls.
Regulation of cellular protein synthesis generates concentration gradients of proteins that affect
the translation of other mRNAs or that determine the pattern formation during early
development.
Different RNAs and functions
RNAs are generally single-stranded. However many RNAs fold back on themselves to form
short regions of double helical structure that are complimentary to each other. As you will see
many times bases in RNA do not follow strict Watson-Crick base pair rules as in DNA and there
is some flexibility. For example the base uracil, which is specific to RNA, pairs with A and also
sometimes with G. Stability of RNA is very poor compared to DNA, and is easily degraded.
Unlike DNA, RNA has a ribose sugar with 2’OH that makes RNA easily susceptible for
cleavage than the deoxyribose sugar present in DNA. Unlike DNA, different kinds of RNAs with
different functions are produced. For example, RNA, like DNA, is the genetic material for many
viruses. However, when these viruses infect their hosts, the RNA can be converted to DNA by an
enzyme called reverse transcriptase and then the viral DNA can integrate with the host
chromosomal DNA. Hence, like DNA that serves as a template for RNA synthesis, RNA also
serves as a template for DNA synthesis in the presence of DNA polymerase-like enzyme called
reverse transcriptase. In fact the RNA component associated with reverse transcriptase-like
enzyme called telomerase is crucially required to replicate the 3’ends of the chromosomes.
Telomerase is an RNA-protein complex.
While telomerase plays an important role in the replication of the DNA at the ends of a
chromosome, DNA polymerases play a role in the replication of the rest of chromosomal DNA.
However DNA polymerases cannot initiate altogether the synthesis of a new DNA chain and can
only elongate a given DNA chain from a 3’-OH group. To solve the DNA replication problems
of both the leading and lagging strands, cells employ small RNA primers in DNA replication.
In the splicing of RNAs, removal of the non-coding regions and joining of the coding regions in
messenger RNAs is accomplished by small nuclear RNA-protein complexes that contain UI, U2,
U4, U5 and U6 RNAs, and different proteins. While ribosomal RNA (rRNA) is an important
component in the structure of ribosomes, the introns in some of the ribosomal RNA, also called
as ribozymes act like protein enzymes in catalyzing their own cleavage from the pre-rRNA
transcripts in the presence of guanosine as a cofactor, without the aid of any proteins. They not
only have a nuclease activity (hydrolysis of RNA) but they also carry a polymerase activity and
are able to join nucleotides through a process called transesterification reaction.
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In fact one of the most important properties of the 16S ribosomal RNA associated with the small
subunit includes the identification of the ‘start codon/site’ in the mRNA template and monitoring
the codon-anticodon interaction. In contrast, the long rRNA transcripts of the large subunit plays
a role in catalyzing the peptide bond formation between adjacent amino acids during the
translocation step in protein synthesis. Thus some rRNAs like introns act on phosphorous
centers and the long rRNA component (23S or 28S rRNA) of the large subunit is associated with
the peptidyl transferase center can act on carbon centers during peptide bond formation.
In the protein synthetic machinery, the messenger RNA (mRNA) template carries the information
for the synthesis of proteins. The nucleotide sequences in mRNA however are identified by small
adaptor molecules called transfer RNAs (tRNAs) that bring specific activated amino acids on to
the template mRNA based on the sequence information in the mRNA. Interestingly, SsrA, kind
of tmRNA (a combination of messenger RNA and transfer RNA with 457 nucleotides) plays an
important role in the translation of broken mRNAs that do not have stop codons. When a stop
codon is not found in broken mRNAs at the 3’ end, then SsrA RNA can be charged with alanine
and binds to a stalled ribosome with the help of elongation factor EF-Tu and GTP. Then this
alanine participates in the peptide bond formation of the incompletely made nascent polypeptide
and ensures the release of the incomplete mRNA. Since SsrA RNA also contains a small open
reading frame that codes for few amino acids and a stop codon, the incomplete polypeptide made
from the broken mRNA is coupled with the small polypeptide by the SsrA RNA before it is
released and the ribosome is recycled.
Different RNAs or their structures play a role in the regulation of gene expression both at the
transcription and translational levels. For example, binding of the signal recognition particle
(SRP) an RNA protein complex, consisting of 7S RNA, to an mRNA that codes for secretory
protein synthesis on the surface of endoplasmic reticulum, halts the synthesis of the secretory
protein and regulates the translational elongation. There are several examples where in
formation of hairpin-like structures in mRNAs can regulate gene expression at transcriptional
and translational level. The structures in RNA serve as sensors for metabolites and uncharged
tRNAs. These RNA regulatory elements are called riboswitches. Another form of RNA called
the guide RNA, has an ability to add or delete a ribonucleotide in an mRNA and can edit RNAs.
RNA editing is a different way to produce different proteins from an mRNA sequence.
In addition to these different forms and functions of RNAs, recent studies lead to the discovery
of micro RNAs (miRNAs) and silencing or small-interfering RNAs (siRNAs) that are
complimentary to certain regions of an mRNA. Because of this complimentarity, these RNAs
can base-pair and inhibit the translation or degrade the mRNAs. MicroRNAs are transcribed in
the nuclei and are processed by RNAse III –like enzyme. The precursor 70 nt miRNAs then folds
into stem-loop structures and are transported to the cytoplasm where they are further processed
to yield mature miRNAs of ~20 nt duplex intermediate by an RNAase-III enzyme called Dicer.
Dicer also processes long double stranded RNA-precursors to produce siRNAs. The processed
miRNAs and siRNAs assemble into different complexes containing various proteins called
miRNP (microRNA protein complex) and RISC (RNA-induced silencing complex) that guide
them to the respective mRNAs where the miRNAs or siRNAs base-pair to the complementary
regions in mRNAs and inhibit the translation or degrade the mRNAs respectively. The only
common protein of both the complexes is Argoanuate and the functions of these proteins are not
clear. Further small double-stranded RNA can also activate protein kinases like PKR that
35
phosphorylate the alpha –subunit of translational initiation factor 2 (eIF2α) and also 2’-5’ A
synthetase that produce 2-5A oligonucleotides which activate RNAse L that degrades RNA as
mentioned under the subheading of interferons.
Many such examples raise the question whether RNA is evolved prior to DNA?
Co-and post-translational modifications of proteins
Proteins, and/or their amino acids, undergo covalent modifications during (co-translational) or
after (post-translational) their synthesis. These modifications play critical role in the synthesis,
stability, structure, function, degradation, and in targeting the proteins to different cellular
locations. The post translational modifications also influence gene expression, and the interaction
of these proteins with other proteins. The covalent modifications of proteins signal the
development, differentiation, degradation, and cell death. Post translational modifications of
proteins can arise due to the addition of certain functional groups to certain amino acids, addition
of a protein or peptide, a change in the chemical nature of amino acid, and protein-processing
that is required for attaining a proper structure or conformation of the protein.
a) Modifications of some of the amino acids: In modifications such as glycosylation,
phosphorylation, ADP-ribosylation, iodination, sulfation, acetylation, alkylation and
formylation, addition of some of the functional groups like sugars, phosphate, ADPribose, methyl, iodine, sulfur, acetyl, methyl, formyl groups respectively can modify
various amino acids in proteins and their functions.
b) Addition of a protein/ peptides, amino acids, or lipids: Examples here include the
addition of ubiquitin protein through an isopeptide bond (as in ubiquitination, see below)
or a ubiquitin-like peptide (SUMOylation); addition of an amino acid such as
polyglutamic acid residues to the C-terminus of proteins (as in glutamylation), and
addition of lipids such as myristic acid (myristoylation) at the N-terminal end, or addition
of prenyl group to the cysteines located near the C-terminal end, and addition of
palmitate to a cysteine at N or C-terminus or in the body of the protein.
c) Change in the chemical nature of amino acids: Conversion of one amino acid to another
as in the case of citrulination, or deamidation where arginine is converted to citruline or
racemization that converts one optical isoform of an amino acid to another as in the
conversion of L-amino acid to D-amino acid
d) Structural changes in proteins that involve addition of S-S- bridges between cysteine
residues and enzymatic or non-enzymatic cleavage of peptide bonds.
A protein may undergo more than one modification (like processing or removal of some amino
acids and addition of disulfides as in insulin). Some of them are reversible and some of them are
irreversible.
Phosphorylation
Phosphorylation is a reversible modification. Addition of a phosphate moiety to the hydroxyl
groups in serine, threonine and tyrosine amino acids can occur in many proteins by respective
kinases. Dephosphorylation is facilitated by protein phosphatases. The conversion of glucose
monomers to the glycogen polymer requires the enzyme glycogen synthase in vertebrates.
Phosphorylation of glycogen synthase generates inactive form of the enzyme. Dephosphorylated
36
form generated by a protein phosphatase is found to be active. Many factors involved in the
initiation and elongation of eukaryotic translation (protein synthesis) and transcription (RNA
synthesis) regulate their activity through phosphorylation-dephosphorylation. For example
phosphorylation of a small portion of the alpha-subunit of heterotrimeric eukaryotic initiation
factor 2 (eIF2α) that occurs in response to diverse stress conditions inhibits general protein
synthesis but upregulates the translation of certain messenger RNAs like ATF4 or GCN4. It is a
stress signal and affects stress-induced gene expression. Phosphorylation of the serine 51 residue
in the alpha-subunit of heterotrimeric eIF2 affects its interaction with other proteins. For
example, it forms a complex with another rate-limiting pentameric initiation factor called eIF2B
and inhibits the ability of eIF2B to recycle inactive eIF2.GDP to eIF2.GTP.
Methylation and Acetylation
The N-terminal tails of H3 and H4 histone proteins (proteins that wrap around the chromosomes)
undergo several reversible modifications such as phosphorylation (as described above),
acetylation and methylation. Methylation occurs on lysine or arginine residues whereas
acetylation occurs on lysine residues. All of them are reversible modifications. Acetyl
transferases and methyl transferases are the enzymes that catalyze the addition of an acetyl group
to a lysine, and a methyl group to arginine or lysine respectively. Histone acetylation has a role
in chromatin assembly and transcription. Acetylated histone proteins are found associated with
regions of chromosomes that are transcriptionally active whereas deacetylated proteins are found
associated with inactive chromosomes thereby suggesting acetylation of histone proteins may
regulate RNA synthesis. In contrast, methylated histone proteins are found associated with both
transcriptionally active and repressed chromosomes. It is likely the function varies depending on
the position and the amino acid that is modified.
Glycation and Glycosylation
Addition of one or several carbohydrate groups occurs in most of the secretory proteins that are
synthesized on the surface of endoplasmic reticulum and are eventually found on the cell surface
of eukaryotes. Generally the cytoplasmic proteins are not glycosylated except in pathological
conditions like diabetes where the blood glucose levels are high. For example, glycosylation of
hemoglobin that occurs non- enzymatically between the C-1 carbon of glucose and the Nterminus of hemoglobin polypeptides is taken as a measure to monitor the severity of diabetes.
Glycation or addition of a glucose non- enzymatically differs from glycosylation. In glycation,
the free amino group in proteins reacts with glucose forming a ketoamine called Amadori
product. The glycated proteins forms cross links with other proteins, leading to structural and
functional modifications. This modification is generally observed in collagen, lens crystalline
and basement membrane proteins during ageing.
In contrast, glycosylation of normal proteins is an enzyme-catalyzed process. Two classes of
glycosylations are identified. These are O-linked and N-linked. In O-linked glycosylation, the
sugars are added to serine or threonine amino acids generally and also to a modified
hydroxylysine as in the case of collagen protein. O-linked glycans are present in the blood group
antigens. In N-linked glycosylation, sugars are added to the amide nitrogen of aspargine. In
addition to these differences, O-linked glycosylation appears to be a post-translational event and
occurs after the synthesis of the protein. In contrast, N-linked glycosylation is a co-translational
event and occurs during the synthesis of proteins and mainly on the endoplasmic reticulum.
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Further, the mechanisms involving the transfer of sugars to the corresponding proteins in the Oand N-linked glycosylations differ. Attachment of sugars to the proteins requires energy input.
In both cases, the sugar is first complexed with a nucleotide (ex; UDP-glucose or galactose or
UMP-glucose; GDP-mannose etc.,) generally in cytosol. But CMP-sialic acid is formed in the
nucleus. In O-linked glycosylation, the activated sugar, or the nucleotide-linked sugar, is then
transferred to a serine or threonine amino acid of a protein by a membrane bound glucosyl
transferase enzyme. Each type of sugar transfer to a protein is catalyzed by a different type of
glycosyl transferase. Glucosyl transferase enzymes not only use nucleotide- linked sugars but
also use lipid- linked sugars and transfer them to the corresponding proteins as in the case of Nlinked glycosylation. N-linked glycosylation is a complex process. Here, the nucleotide sugar is
first transferred to a lipid intermediate called dolichol phosphate by a glucosyl transferase
enzyme before it is transferred to the amide nitrogen of aspargine of a glycosylated protein that is
placed in a sequence Asn-X (any amino acid)-Ser/thr-Protein by an ologosachharyl transferase.
However, some of these residues are processed or removed when the proteins are processed in
the lumen of the endoplasmic reticulum or by glycosidases. An analysis of oligosaccharides of
N-linked-glycosylated proteins reveals however that mostly they all contain a core
pentasaccharide structure that is generally (3 mannose and two N-acetylgucosamine residues).
The first sugar N-acetylglucosamine is the one linked to the aspargine residue of the protein. In
addition to the core oligosaccharide residues, many N-linked glycosylated proteins have other
sugar residues added to the protein.
Further, some of these sugar residues (like the mannose residues linked to asparagines) of
secretory proteins that are destined to reach lysosomes are also found phosphorylated. Examples
of N-linked glycosylation include ribonuclease B, human IgG. Glycosylation plays a role in
providing a correct charge, increases the solubility, stability and conformation of proteins.
Different types of glycosylation regulate the targeting of proteins to different cellular locations or
organelles.
Oxidation-reduction
Many of the cytoplasmic proteins in higher eukaryotes maintain their –SH groups because of a
higher reducing potential in the cytoplasm and may not use these –SH groups to form disulfide
bonds as a means to stabilize their structure. In contrast, several secretory proteins like
immunoglobulins and hormones (like insulin) or digestive enzymes carry disulfide bonds that are
formed between two cysteine residues (Cys-S-S-Cys) during or soon after their synthesis. These
disulfide bridges are required to maintain the secondary structure of the protein. Oxidative
modification of proteins can also occur by oxygen free radicals, metal-catalyzed oxidation
systems and mixed function oxidation systems. These systems can oxidize some amino acids like
proline, arginine and lysine to carbonyl derivatives. The amount of carbonyl content of proteins
has been used to estimate protein oxidation during ageing.
ADP-ribosylation
It involves the addition of ADP-ribosyl moiety to an arginine or modified histidine amino acid
residue in the presence of an enzyme ADP-ribosyl transferase. The ADP-ribosyl moiety comes
from NAD+. A modified histidine residue in elongation factor 2 (EF2) in eukaryotes can be
ADP-ribosylated in the presence of diphtheria toxin that contains ADP-ribosyl transferase
activity. ADP-ribosylated EF2 inhibits protein synthesis. Cholera toxin also contains similar
38
enzymatic activity and is used to demonstrate that ADP ribosylation of the Gα subunit of the
trimeric G-protein can block the GTPase activity associated with the protein and can convert the
α-subunit into an inrreversible activator of adenylate cyclase, the enzyme that produces cyclic
AMP from ATP.
Iodination
The synthesis of thyroid hormones, mainly thyroxine (T4) and triiodothyronine (T3) by thyroid
gland, involves iodination of the tyrosine ring. The iodide ion required for this process is
obtained from blood serum. Deficiency of iodine results in goiter, a disease in which thyroid
gland grows abnormally. Thyroid hormones affect the expression of several genes that are
involved in various metabolic processes.
Formylation
Formylated methionine is the first amino acid to be incorporated in all the bacterial proteins.
Transformylase enzyme modifies methionine that is already attached to the initiator tRNA in
prokaryotes by transferring a formyl group from N-10 formyl tetrahydrofolate. Deformylase
enzyme removes the formyl group during the elongation step of protein synthesis. An amino
peptidase enzyme also removes the first methionine from many proteins. Formylation favors
selection of formylated Met-tRNAfMet by IF2 protein factor in prokaryotes, blocks the joining of
the f-Met-tRNAfMet to the elongation factor EF-Tu.
Lipoylation
This refers to proteins with covalently attached lipids such as myristic acid, a fourteen –carbon
saturated fatty acid (C14: 0), palmitic acid, a saturated sixteen –carbon fatty acid (C16: 0), farnesyl,
fifteen carbon isoprenoid, and geranylgeranyl, a twenty carbon isoprenoid etc., The
modifications are called as N-myristoylation, palmotoylation and prenylation respectively. In Nmyristoylation, myristc acd is bound to the N-terminal glycine of a variety of proteins by an
amide bond. Myristoyl-CoA:protein:N-myristoyl transferase enzyme acylates the nascent
polypeptides after cellular methionyl aminopeptidases remove the first or initiation methionine.
Substrates for myristoylation include kinases like protein kinaseA, phosphatases like calcineurin
B, transmembrane proteins like the alpha-subunit of heterotrimeric G-protein and a retroviral
polyprotein gag coded by HIV. Prenylation involves the attachment of a prenyl group (farnesyl
diphosphate or geranylgeranyl diphosphate) to the cysteines located near the C-terminus of a
protein by a thioester bond whereas palimitoylation involves the addition of a palimitate to a
cysteine by protein palimitoyl transferase to the N- or C-terminus of the protein and can also
occur in the body of the protein. Most Ras proteins are substrates for palmitoylation. The γsubunit of G-protein coupled receptor in the retina is a substrate for prenylation. Palmitoylation
can also occur at times nonenzymatically. Palimitoylation increases the hydrophobicity of a
protein molecule so that its association with cellular membranes can occur whereas simple
myristoylation or prenylation is not sufficient for a protein to interact with the plasmamebrane.
Addition of another lipid molecule in the case of myristoylation or methylation of the carboxyl
groups of cysteine in prenylation may enhance their affinity for membranes.
39
Protein Splicing
Like RNA splicing and maturation, most proteins produced soon after their synthesis lose one or
several of the amino acids to form a mature and functional protein with a proper structure. The
removal of the amino acids in proteins can occur in the presence and absence of enzymes. Many
proteins contain sequences called inteins that have an endonuclease domain inserted into the
protein and exteins. Inteins are excised in the final protein and the mature host protein contains
exteins. The inteins are comparable to introns and the exteins are to exons in RNA. The removal
of inteins is autocatalytic and does not involve any enzymes. Most inteins have a central
conserved aminoacid sequence (LAGLIDADG) of an endonuclease domain and many inteins
also do not have such an endonuclease domain. The DNA sequences responsible for protein
inteins can be modified so that the protein splicing can be controlled at the will of the researcher.
Protein processing
Protein processing can also occur by different proteases. Processing facilitates the generation of
biologically active protein. Many of the digestive enzymes and hormones are synthesized as prepro proteins containing several additional amino acids that are not found in the mature and
processed protein. These additional amino acids are removed by proteases. Examples include
many of the pancreatic proteases like trypsin, elastase, enteropeptidase and carboxypeptidase.
Most of these enzymes are made on the rough surface of endoplasmic reticulum and are then
moved into the lumen of the endoplasmic reticulum. Here the N-terminal signal sequence
characteristic of the secretory proteins is cleaved by a signal peptidase. Afterwards, some of
these proteins are further processed. For example the conversion of chymotrypsinogen to active
π chymotrypsin is done by trypsin that cleaves peptide between arginine 15 and isoleucine 16 at
the N-terminus of the protein. This is further followed by autocatalytic processing of πchymotrypsin that removes amino acid residues 14-15 and 47-148 from the molecule to produce
final α-chymotrypsin. The cleavage of a hexapeptide from the N-terminus end of trypsinogen in
duodenal cells to active trypsin occurs by enteropeptidase. Active trypsin also catalyzes inactive
trypsinogen to active trypsin. Another important example involves a cascade of proteolytic
activation of specific proteases that will process prothrombin to thrombin which in turn catalyzes
the conversion of fibrinogen, a serine protease, to fibrin during blood clotting. Thrombin cleaves
mainly a bond between Arg-Gly. Similarly in programmed cell death, cysteinyl aspartate
proteases called caspases get activated and act on procaspases and convert them to active
caspases. The proteases identify specific amino acid sequences in proteins. Active caspases in
turn cleave several protein substrates and modify their activities.
Other modifications
Other covalent modifications include alkylation (addition of methyl or ethyl groups to lysine or
arginine amino acids), glutamylation (covalent linkage of glutamic acid residues to the cterminus of tubulin) and sulfation, addition of sulfate group to tyrosine). Further, often there is an
interconversion of optical isoforms of amino acids (L-amino acid to D-amino acid) called
racemization and conversion of a neutral amide group to an acidic group by deamidation.
Spontaneous deamidation of the amino acids of glutamine and aspargine occurs in several
proteins during ageing.
40
Ubiquitnation
Many proteins, prior to degradation by a proteaosome, are coupled or conjugated enzymatically
with a protein called ubiquitin (Ub). Ubiquitin tag to a protein signals for degradation. This type
of covalent modification is different from other modifications of amino acids that have been
described above. Here the target or substrate protein is attached with another polypeptide like
ubiquitin. Recent studies indicate that many small polypeptides that are distinct from ubiquitin
but related to ubiquitin are tagged enzymatically to target molecules in different biological
processes such as DNA repair, autophagy and signal transduction. Ubiquitination of proteins
provides large and better chemical surface. The process requires three to four enzymes. The
sequence of events that occur during ubiquitination are as follows: In step 1, a deubiquitinating
enzyme processes the precursor ubiquitin protein to generate ubiquitin that contains Gly-Gly
sequence at the C–terminus. In step 2, activation of ubiquitin occurs by enzyme 1 and ATP. In
the activation step, C-terminus of ubiquitin is adenylated by enzyme 1. Adenylated ubquitin is
then linked to the enzyme through a high-energy thioester bond. In step 3, activated ubiquitin is
passed to cysteinyl group of the enzyme 2 called ubiquitin-conjugating enzyme (Fig. 19). In
some cases, enzyme 3, ubiquitin-protein ligase, combines ubiquitin and the target protein
(substrate) through an amide (isopeptide) bond. Otherwise enzyme2-conjugated ubiquitin is
sufficient for modification of the substrate protein in many cases.
Ubiquitin (Ub) is covalently attached to other proteins (target protein) through an isopeptide
bond. The isopeptide bond occurs between the carboxy-terminal glycine of Ub and the epsilonamino group of lysine in the target protein (Fig. 19). Target protein could be another ub, or
ubiquitin-like protein (SUMO or small Ub-like modifier, autophagy-12, interferon stimulated
gene of 15 kDa) or structurally a different protein substrate. Depending on the number of
ubiquitin molecules attached, the function may differ. For example, mono- and di- ubiquitination
are implicated in endocytosis, and a chain containing minimum four molecules of Ub may be
required for efficient proteasomal degradation. Also the lysine residues in Ub that is involved in
the linkage or in the formation of isopeptide bond may play a role. Lysine (K) 48 –linked chains
are involved in proteasomal degradation and K63 linkage may be involved other processes like
endocytosis or DNA-repair.
Fig. 19: Ubiquitination
41
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