Download Nucleic Acids - Farmasi Unand

Reference: Gareth Thomas
Week 11, 12,13
prof. aza
1. Introduction
• The nucleic acids are the
compounds that are responsible for
the storage and transmission of the
genetic information that controls
the growth, function and
reproduction of all types of cells.
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• They are classified into two general
types: the deoxyribonucleic acids
(DNA), whose structures contain
the sugar residue β-D-deoxyribose;
and the ribonucleic acids (RNA),
whose structures contain the sugar
residue β -D-ribose (Figure 10.1).
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Figure 1. The structures of
β -D-deoxyribose and β -D-ribose.
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nucleotide consists of a purine or
pyrimidine base
• Both types of nucleic acids are polymers
based on a repeating structural unit
known as a nucleotide (Figure 10.2).
These nucleotides form long single-chain
polymer molecules in both DNA and
RNA.
• Each nucleotide consists of a purine or
pyrimidine base bonded to the 1’ carbon
atom of a sugar residue by a β -Nglycosidic link (Figure 10.3).
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• These base-sugar subunits, which
are known as nucleosides, are linked
through the 3’ and 5’ carbons of
their sugar residues by phosphate
units to form the polymer chain.
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Figure 2. The general structures of
(a) nucleotides and (b) a schematic
representation of a section of a
nucleic acid chain.
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Figure 3. Examples of the structures of some
of the nucleosides found in RNA. The β -Nglycosidic link is shaded The corresponding
nucleosides in DNA are based on deoxyribose
and use the same name but with the prefix
deoxy.
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2. Deoxyribonucleic Acids (DNA)
• DNA occurs in the nuclei of cells in
the form of a very compact DNA
protein complex called chromatin.
The protein in chromatin consists
mainly of histones, a family of
relatively small positively charged
proteins.
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• The DNA is coiled twice around as
octomer of histone molecules with a
ninth histone molecule attached to
the exterior of these mini coils to
form a structure like a row of
heads spaced along a string (Figure
10.4).
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• This ‘string of beads’ is coiled and
twisted into compact structures known
as miniband units, which form the basis
of the structures of chromosomes.
Chromosomes are the structures that
form duplicates during cell division in
order to transfer the genetic
information of the old cell to the two
new cells.
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2.1 Structure
DNA molecules are large with relative
molecular masses up to one trillion. The
principal bases found in their structures
are adenine (A), thymine (T), guanine (G)
and cytosine (C), although derivatives of
these bases are found in some DNA
molecules (Figure 10.5). Those bases
with an oxygen function have been
shown to exist in their keto form.
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• Figure 4. The ‘string of heads’ structure
of chromatin. The DNA strand is round
twice around each histone octomer. A
ninth histone molecule is bound to the
exterior surface of the coil.
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DNA-binding proteins
Interaction of DNA
with histones
(shown in white,
top). These
proteins' basic
amino acids (below
left, blue) bind to
the acidic
phosphate groups
on DNA (below
right, red).
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Figure 5. The purine and pyrimidine bases
found in DNA. The numbering is the same
or each type of ring system.
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Structures of the four bases found in DNA and the
nucleotide adenosine monophosphate.
Adenine
Guanine
Adenosine monophosphate
Thymine
Cytosine
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• Chargaff showed that the molar
ratios of adenine to thymine and
guanine to cytosine are always
approximately 1: 1 in any DNA
structure although the ratio of
adenine to guanine varies according
to the species from which the DNA
is obtained.
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• This and other experimental
observations lead Crick and Watson in
1953 to propose that the threedimensional structure of DNA consisted
of two single molecule polymer chains
held together in the form of a double
helix by hydrogen bonding between the
same pairs of bases, namely:
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• the adenine- thymine and cytosine-
guanine base pairs (Figure 10.6).
These pairs of bases, which are
referred to as complementary base
pairs, form the internal structure
of the helix.
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• They are hydrogen bonded in such
a manner that their flat structures
lie parallel to one another across
the inside of the helix. The two
polymer chains forming the helix
are aligned in opposite directions.
In other words, at the ends of the
structure one chain has a free 3’OH group and the other chain has a
free 5’-OH group.
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• X-ray diffraction studies have since
confirmed that this is the basic three
dimensional shape of the polymer chains
of the β -DNA, the natural form of
DNA.
• This form of DNA has about ten bases
per turn of the helix.
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• Its outer surface has two grooves
known as the minor and major grooves,
respectively, which act as the binding
sites for many ligands. Two other forms
of DNA, the A and Z forms, have also
been identified but it is not certain if
these forms occur naturally in living
cells.
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• Electron microscopy has shown that the
double helical chain of DNA is folded,
twisted and coiled into quite compact
shapes. A number of DNA structures
are cyclic and these compounds are also
coiled and twisted into specific shapes.
These shapes are referred to as
supercoils, supertwists and superhelices
as appropriate.
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The two strands
of DNA are held
together by
hydrogen bonds
between bases.
The sugars in
the backbone
are shown in
light blue.
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• Figure 10.6. The double helical
structure of B-DNA. Interchanging of
either the bases of a base pair and/or
base pair with base pair does not affect
the geometry of this structure.
Reproduced from G. Thomas. (Chemistry for
Pharmacy and the Life Sciences including Pharmacology and
Biomedical Science, I996, by per mission of Prentice Hall, a
Pearson Education Company.
prof. aza
3. The General Functions of DNA
The DNA found in the nuclei of cells has
three functions:
(i) to act as a repository for the genetic
information required by a cell to
reproduce that cell:
(ii) to reproduce itself in order to
maintain the genetic pool when cells
divide;
(iii) to supply the information that the
cell requires to manufacture specific
proteins.
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• Genetic information is stored in a form
known as genes by the DNA found in the
nucleus of a cell (see section 10.4).
• The duplication of DNA is known as
replication. It results in the formation
of two identical DNA molecules that
carry the same genetic information
from the original cell
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• to the two new cells that are formed
when a cell divides (see section 10.5).
• The function of DNA in protein
synthesis is to act as a template for the
production of the various RNA
molecules necessary to produce a
specific protein (see section 6)
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Figure 10.7. A schematic
representation of the gene for
the β -subunit of haemoglobin.
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4. Genes
• Each species has its own internal and
external characteristics. These
characteristics are determined by the
information stored and supplied by the
DNA in the nuclei of its cells.
• This information is carried in the form
of a code based on the consecutive
sequences of bases found in sections of
the DNA structure (see section 5).
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• This code controls the production of
the peptides and proteins required by
the body.
• The sequence of bases that act as the
code for the production of one specific
peptide or protein molecule is known as
a gene.
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Changing the sequence of the bases effect on
the external or internal characteristics of an
individual
• Genes can normally contain from several
hundred to 2000 bases. Changing the
sequence of the bases in a gene by
adding, subtracting or changing one or
more bases may cause a change in the
structure of that protein with a
subsequent knock-on effect on the
external or internal characteristics of
an individual.
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• For example, an individual may have
brown instead of blue eyes or their
insulin production may be inhibited,
which could result in that individual
suffering from diabetes.
• A number of medical conditions have
been attributed to either the absence
of a gene or the presence of a
degenerate or faulty gene in which one
or more of the bases in the sequence
have been changed.
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• In simple organisms, such as bacteria,
genetic information is usually stored in a
continous sequence of DNA bases.
• However, in higher organisms the bases
forming a particular gene may occur in a
number of separate sections known as
exons, separated by sections of DNA
that do not appear to be a code for any
process.
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• These non-coding sections are
referred to as introns. For
example, the gene responsible for
the β-subunit of haemoglobin
consists of 990 bases. These bases
occur as three exons separated by
two introns (Figure 10.7).
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• The complete set of genes that
contain all the hereditary
information of a particular species
is called a genome.
• The Human Genome Project.
initiated in 1990, sets out to
identify all the genes that occur in
human chromosomes and also the
sequence of bases in these genes.
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• This will create an index that can
be used to locate the genes
responsible for particular medical
conditions. For example, the gene in
region q31 of chromosome 7 is
responsible for the protein that
controls the flow of chloride ions
through the membranes in the
lungs.
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• The changing of about three bases
in exon number 10 gives a
degenerate gene that is known to
be responsible for causing cystic
fibrosis in a large number of cases.
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Figure 10.8. A schematic representation of the
replication of DNA. The arrows show the direction
of growth of the leading and lagging strands.
Reproduced from G. Thomas, Chemistry for Pharmacy and the Life Sciences including Pharmacology and Biomedical
Science, 1996, by permission of Prentice Hall, a Pearson Education Company.
prof. aza
• Figure 10.8. A schematic
representation of the replication of
DNA. The arrows show the
direction of growth of the leading
and lagging strands.
• Reproduced from G. Thomas, Chemistry for Pharmacy
and the Life Sciences including Pharmacology and
Biomedical Science, 1996, by permission of Prentice
Hall, a Pearson Education Company.
prof. aza
DNA replication. The
double helix (blue) is
unwound by a helicase.
Next, DNA polymerase III
(green) produces the
leading strand copy (red).
A DNA polymerase I
molecule (green) binds to
the lagging strand. This
enzyme makes
discontinuous segments
(called Okazaki fragments)
before DNA ligase (violet)
joins them together.
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5. Replication
• Replication is believed to start with the
unwinding of a section of the double
helix (Figure 10.8).
• Unwinding may start at the end or more
commonly in a central section of the
DNA helix. It is initiated by the binding
of the DNA to specific receptor
proteins that have been activated by
the appropriate first messenger (see
section 8.4).
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• The separated strands of the DNA act
as templates for the formation of a new
daughter strand.
• Individual nucleotides, which are
synthesised in the cell by a complex
route, bind by hydrogen bonding
between the bases to the
complementary parent nucleotides.
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• This hydrogen bonding is specific:
only the complementary base pairs
can hydrogen bond.
• In other words, the hydrogen
bonding can only be between either
thymine and adenine or cytosine and
guanine.
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• This means that the new daughter
strand is an exact replica of the
original DNA strand bound to the
parent strand.
• Consequently, replication will
produce two identical DNA
molecules.
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• As the nucleotides hydrogen bond to
the parent strand they are linked to the
adjacent nucleotide, which is already
hydrogen bonded to the parent strand,
by the action of enzymes known as DNA
polymerases.
• As the daughter strands grow, the DNA
helix continues to unwind.
• However, both daughter strands are
formed at the same time in the 5’ to the
3’ direction.
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• This means that the growth of the
daughter strand that starts at the 3’
end of the parent strand can continue
smoothly as the DNA helix continues to
unwind.
• This strand is known the leading strand.
However, this smooth growth is riot
possible for the daughter strand that
started from the 5’ of the parent
strand.
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• This strand, known as the lagging
strand, is formed in a series of sections,
each of which still grows in the 5’ to 3’
direction.
• These sections. which are known as
Okazaki fragments after their
discoverer, are joined together by the
enzyme DNA ligase to form the second
daughter strand.
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• Replication, which starts at the end of
a DNA helix, continues until the entire
structure has been duplicated.
• The same result is obtained when
replication starts at the centre of a
DNA helix.
• In this case, unwinding continues in both
directions until the complete molecule is
duplicated. This latter situation is more
common.
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• DNA replication occurs when cell
division is imminent. At the same time,
new histones are synthesised.
• This results in a thickening of the
chromatin filaments into chromosomes
(see section 2). These rod-like
structures can be stained and are large
enough to be seen under a microscope.
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6. Ribonucleic Acids (RNA)
• Ribonucleic acids are found in both the
nucleus and the cytoplasm. In the
cytoplasm RNA is located mainly in small
spherical organelles known as ribosome.
These consist of about 65% RNA and
35% protein.
• Ribonucleic acids are classified
according to their general role in
protein synthesis as: messenger RNA
(mRNA): transfer RNA (tRNA): and
ribosomal RNA (rRNA).
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• Messenger RNA informs the ribosome
as to what amino acids are required and
their order in the protein, that is, they
carry the genetic information necessary
to produce a specific protein.
• This type of RNA is synthesised as
required and once its message has been
delivered it is decomposed.
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Figure 10.9. (a) The general structure ol a section of
an RNA polymer chain. (b) The hydrogen bonding
between uracil and adenine. Reproduced from
G.’Thomas, Chemistry to Pharmacy and the Life
Science including Pharmacology including Biomedical
Science, 1996, by permission of Prentice Hall, a
Pearson Education Company.
prof. aza
• Figure 10.9. (a) The general structure of
a section of an RNA polymer chain. (b)
The hydrogen bonding between uracil
and adenine. Reproduced from G.’Thomas, Chemistry to
Pharmacy and the Life Science including Pharmacology including
Biomedical Science, 1996, by permission of Prentice Hall, a
Pearson Education Company.
prof. aza
Figure 10.10. A schematic representation of a
transcription process. Reproduced from
G.’Thomas, Chemistry to Pharmacy and the Life
Science including Pharmacology including
Biomedical Science, 1996, by permission of
Prentice Hall, a Pearson Education Company.
prof. aza
• The structures of RNA molecules
consist of a single polymer chain of
nucleotides with the same bases as
DNA, with the exception of thymine,
which is replaced by uracil ( Figure 9).
• These chains often contain singlestranded loops separated by short
sections of a distorted double helix
(Figure 11). These structures are known
as hairpin loops.
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• All types of RNA are formed from DNA
by a process known as transcription. It
is thought that the DNA unwinds and
the RNA molecule is formed in the 5’ to
3’ direction.
• It proceeds smoothly. with the 3’ end of
the new strand bonding to the 5’ end of
the next nucleotide (Figure 10.10).
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• This bonding is catalysed by enzymes
known as RNA polymerases.
• The sequence of bases in the new RNA
strand is controlled by the sequence of
bases in the parent DNA strand.
• In this way DNA controls the genetic
information being transcribed into the
RNA molecule.
• The strands of DNA also contain start
and stop signals, which control the size
of the RNA molecule produced.
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• These signals are in the form of
specific sequences of bases.
• It is believed that the enzyme rho
factor could be involved in the
termination of the synthesis and the
release of some RNA molecules from
the parent DNA strand. However, in
many cases there is no evidence that
this enzyme is involved in the release of
the RNA molecule.
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• The RNA produced within the
nucleus by transcription is known as
heterogeneous nuclear RNA
(hnRNA), premessenger RNA (premRNA) or primary transcript RNA (
ptRNA).
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• Since the DNA gene from which it is
produced contains both exons and
introns, the hnRNA will also contain its
genetic information in the form of a
series of exons and introns
complementary to those of its parent
gene.
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7. Messenger RNA (mRNA)
• mRNA carries the genetic message from
the DNA in the nucleus to a ribosome.
This message instructs the ribosome to
synthesise a specific protein.
• mRNA is believed to be produced in the
nucleus from hnRNA by removal of the
introns and the splicing together of the
remaining exons into a continuous
genetic message,
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• the process being catalysed by
specialised enzymes. The net result is a
smaller mRNA molecule with a
continuous sequence of bases that are
complementary to the gene’s exons, this
mRNA now leaves the nucleus and
carries its message in the form of a
code to a ribosome.
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all protein synthesis starts with methionine
• The code carried by mRNA was broken
in the 1960s by Nirenberg and other
workers. These workers demonstrated
that each naturally occurring amino acid
had a DNA code that consisted of a
sequence of three consecutive bases
known as a codon and that an amino acid
could have several different codons
(Table 10.1),
• In addition, three of the codons are
stop signals which instruct the ribosome
to stop protein synthesis.
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• Furthermore, the codon that initiates
the synthesis is always AUG, which is
also the codon for methionine.
Consequently, all protein synthesis
starts with methionine.
However, few completed proteins have a
terminal methionine because this
residue is normally removed before the
peptide chain is complete.
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• Moreover, methionine can still be
incorporated in a peptide chain
because there are two different
tRNAs that transfer methionine to
the ribosome (see section 8).
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all living matter using the same genetic code for
protein synthesis
• One is specific for the transfer of
the initial methionine whereas the
other will only deliver methionine to
the developing peptide chain, By
convention, the three letters of
codon triplets are normally written
with their 5’ ends on the left and
their 3’ ends on the right.
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• The mRNA’s codon code is known as
the genetic code, Its use is
universal, all living matter using the
same genetic code for protein
synthesis.
• This suggests that all living matter
must have originated from the same
source and is strong evidence for
Darwin’s theory of evolution.
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• Figure .11. The general structures of
tRNA. (a) The two-dimensional
cloverleaf representation showing some
of the invariable nucleotides that occur
in the same positions in most tRNA
molecules and (b) the three- dimensional
L shape (From CHEMISTRY, by Linus Pauling and
Peter Pauling. Copyright © 1975 by Linus Pauling and
Peter Paling. Used with permission of W. H. Freeman
and Company)
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8. Transfer RNA (tRNA)
• tRNAs are also believed to be formed in
the nucleus from the hnRNA.
• They are relatively small molecules that
usually contain from 73 to 94
nucleotides in a single strand. Some of
these nucleotides may contain
derivatives of the principal bases, such
as 2’-O-methylguanosine (0MG) and
inosine (I).
• The strand of tRNA is usually folded
into a three-dimensional L shape.
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• This structure, which consists of
several loops, is held in this shape
by hydrogen bonding between
complementary base pairs in the
stem sections of these loops and
also by hydrogen bonding between
bases in different loops.
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Figure .11. The general structures of tRNA. (a) The two-dimensional
cloverleaf representation showing some of the invariable nucleotides
that occur in the same positions in most tRNA molecules and (b) the
three- dimensional L shape (From CHEMISTRY, by Linus Pauling and Peter Pauling.
Copyright © 1975 by Linus Pauling and Peter Paling. Used with permission of W. H. Freeman and
Company)
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• Figure .11. The general structures of
tRNA. (a) The two-dimensional
cloverleaf representation showing some
of the invariable nucleotides that occur
in the same positions in most tRNA
molecules and (b) the three- dimensional
L shape (From CHEMISTRY, by Linus Pauling and
Peter Pauling. Copyright © 1975 by Linus Pauling and
Peter Paling. Used with permission of W. H. Freeman
and Company)
prof. aza
• This results in the formation of
sections of double helical structures.
• However, the structures of most
tRNAs are represented in two
dimensions as a cloverleaf (Figure
11).
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• tRNA molecules carry amino acid
residues from the cell’s amino acid
pool to the mRNA attached to the
ribosome.
• The amino acid residue is attached
through an ester linkage to
ribosome residue at the 3’ terminal
of the tRNA strand, which almost
invariably has the sequence CCA.
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• This sequence plus a fourth nucleotide
projects beyond the double helix of the
stem. Each type of amino acid can only
be transported by its own specific
tRNA molecule. In other words a tRNA
that carries serine residues will not
transport alanine residues. In other
word, some amino acids can be carried
by several different tRNA molecules
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• The tRNA recognises the point on the
mRNA where it has to deliver its amino
acid through the use of a group of three
bases known as an anticodon.
• This anticodon is a sequence of three
bases found on one of the loops of the
tRNA (Figure 11).
• The anticodon can only form base with
the complementary codon in the mRNA.
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• Consequently, the tRNA will only
hydrogen bond to the region of the
mRNA that has the correct codon,
which means its amino acid can only
be delivered to a specific point on
the mRNA.
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• For example, a tRNA molecule with the
anticodon CGA will only transport its
alanine residue to a GCU codon on the
mRNA.
• Furthermore, this mechanism will also
control the order in which amino acid
residues are added to the growing
protein.
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9. Ribosomal RNA (rRNA)
• Ribosomes contain about 35% protein
and 65% rRNA.
• Their structures are complex and have
not yet been fully elucidated.
• However, they have been found to
consist of two Sections that are
referred to as the large and small
subunits.
• Each of these subunits contains protein
and rRNA
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• In Eschericia coli the small subunit has
been shown to contain a 1542-nucleotide
rRNA molecule whereas the large
contains two rRNA molecules of 120
(Figure 12) and 2094 nucleotides,
respectively.
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• Experimental evidence suggests that
rRNA molecules have structures that
consist of a single strand of nucleotides
whose sequence varies considerably
from species to species.
• The strand is folded and twisted to
form a series of single-stranded loops
separated by sections of double helix
(Figure 12).
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Figure 10.12. The proposed sequence of nucleotides in the
120-nucleotide subunit found in Escherichia coli ribosome
showing the single-stranded loops and the double helical
structures. (Reprinted, with permission, from the Annual Review of Biochemistry, volume 53 ©
I984 by Annual Reviews. www.Annual Reviews.org).
prof. aza
• The double helical segments are
believed to be formed by hydrogen
bonding between complementary base
pairs.
• The general pattern of loops and helixes
is very similar between species even
though the sequence of nucleotides are
different
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• However, little is known about the
three-dimensional structures of
rRNA molecules and their
interactions with the proteins
found in the ribosome.
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10. Protein Synthesis
• Protein synthesis starts from the
N-terminal of the protein.
• It proceeds in the 5’ to 3’ direction
along the mRNA and may be divided
into four mayor stages. namely:
activation: initiation: elongation:
and termination.
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Activation
• Activation is the formation of the
tRNA amino acid complex.
• Initiation is the binding of the
mRNA to the ribosome and the
activation of the ribosome.
Elongation is the formation of the
protein.
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• Termination is the ending of the protein
synthesis and its release from the
ribosome.
• All these processes normally require the
participation of protein catalysts, known
as factors, as well as other proteins
whose function is not always known.
• GTP and sometimes ATP act as sources
of energy for the processes.
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10.1 Activation
It is believed that the amino acids from the cellular pool react with
ATP to form an active amino acid-AMP complex. This complex reacts
with the specific tRNA for the amino acid. the reaction being
catalysed by a synthese that is specific for that amino acid.
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• It is believed that the amino acids ( AA)
from the cellular pool react with ATP to
form an active amino acid-AMP complex
(AA-AMP).
• This complex reacts with the specific
tRNA for the amino acid, the reaction
being catalysed by a synthase that is
specific for that amino acid.
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Figure 13. A schematic representation of
the initiation of protein synthesis.
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2. Initiation
• The general mechanism of initiation is
well documented but the liner details
are still not known.
• It is thought that it starts with the two
subunits of the ribosome separating and
the binding of the mRNA to the smaller
subunit.
• Protein synthesis then starts by the
attachment of a methionine-tRNA
complex to the mRNA so that it forms
the N-terminal of the new protein.
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• Methionine is always the first amino
acid in all protein synthesis because its
tRNA anticodon is also the signal for
the ribosome system to start protein
synthesis.
• Because the anticodon for methionine
tRNA is UAC, this synthesis will start
at the AUG codon of the mRNA.
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• This codon is usually found within
the first 30 nucleotides of the
mRNA.
• However, few proteins have an Nterminal methionine because once
protein synthesis has started the
methionine is usually removed by
hydrolysis.
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• As soon as the methionine-tRNA has
bound to the mRNA the larger
ribosomal subunit is believed to bind to
the smaller subunit so that the mRNA is
sandwiched between the two subunits
(Figure 13).
• This large subunit is believed to have
three binding sites called the P
(peptidyl), A (acceptor) and E (exit)
sites.
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• It attaches itself to the smaller
subunit so that its P site is aligned
with the methionine- tRNA complex
bound to the mRNA.
• This P site is where the growing
protein will be bound to the
ribosome.
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• The A site, which is thought to be
adjacent to the P site, is where the
next amino acid-tRNA complex binds to
the ribosome so that its amino acid can
be attached to the peptide chain.
• The E site is where the discharged
tRNA is transiently bound before it
leaves the ribosome.
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• This large subunit is believed to
have three binding sites called the
P (peptidyl), A (acceptor) and E
(exit) sites.
• It attaches itself to the smaller
subunit so that its P site is aligned
with the methionine-tRNA complex
bound to the mRNA.
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• This P site is where the growing
protein will be bound to the
ribosome.
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• The A site, which is thought to be
adjacent to the P site, is where the
next amino acid-tRNA complex binds to
the ribosome so that its amino acid can
be attached to the peptide chain.
• The E site is where the discharged
tRNA is transiently bound before it
leaves the ribosome
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3.
Elongation
• Elongation is the formation of
the peptide chain of the protein
by a stepwise repetitive process.
• A great deal is known about the
nature of this process but its
exact mechanism is still not fully
understood.
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• The process of elongation is best
explained by the use of a
hypothetical example.
• Suppose that the sequence of
codons, including the start codon, is
AUGUUGGCUGGA.. etc
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• The elongation process starts with
the methionine-tRNA bound to the
AUG codon of the mRNA (Figure
14).
• Because the second codon is UUG
the second amino acid in the
polypetide chain will be leucine.
prof. aza
• This amino acid is transported by a
tRNA molecule with the anticodon
AAC because this is the only
anticodon that matches the UUG
codon on the mRNA strand.
• The leucine- tRNA complex ‘docks’
on the UUG codon of the mRNA and
binds to the A site.
prof. aza
• This docking and binding is believed to
involve ribosome proteins, referred to
as elongation factors, and energy
supplied by the hydrolysis of guanosine
triphosphate (GTP) to guanosine
diphosphate (GDP).
• Once the leucine-tRNA has occupied the
A site the methionin is linked to the
leucine by means of a peptide link whose
carbonyl group originates from the
methionine.
prof. aza
• This reaction is catalysed by the
appropriate transferase.
• It leaves the tRNA on the P site
empty and produces an (NH2)-MetLeu-tRNA complex at the A site.
prof. aza
• The empty tRNA is discharged
through the E site and at the same
time the complete ribosome moves
along the mRNA in the 5’ to 3’
direction so that the dipeptidetRNA complex moves from the A
site to the P site.
prof. aza
• This process is known as
translocation.
• It is poorly understood but it
leaves the A site empty and able to
receive the next amino acid tRNA
complex.
• The whole process is then repeated
in order to add the next amino acid
residue to peptide chain.
prof. aza
• Because the next mRNA codon in our
hypothetical example is (GCU) this
amino acid will be alanine (see Table 10.
l). Subsequent amino acids are added in
a similar way, the sequence of amino
acid residues in the chain being control
led by the order of the codons in the
mRNA.
prof. aza
• It is poorly understood but it
leaves the A site empty and able to
receive the next amino acid tRNA
complex. The whole process is then
repeated in order to add the next
amino acid residue to peptide chain
.
prof. aza
• Figure 13. A diagrammatic representation of the
process of elongation in protein synthesis
prof. aza
10.4 Termination
• The elongation process continues
until a stop codon is reached.
• This codon cannot accept an amino
acid-tRNA complex and so the
synthesis stops.
• At this point the peptide-tRN chain
occupies a P site and the A site is
empty.
prof. aza
• The stop codon of the mRNA is
recognised by proteins know as
release factors, which promote the
release of the protein from the
ribosome.
prof. aza
• The mechanism by which this
happens is not fully understood but
they are believed convert the
transferase responsible for peptide
synthesis into a hydrolase, which
catalyses hydrolysis of the ester
group linking the polypeptide to its
tRNA.
prof. aza
• Once released, the protein is
folded into its characteristic
shape. often under the direction
of molecular chaperone protein.
prof. aza
11 Protein Synthesis in Prokaryotic and
Eukaryotic Cells
• The general sequence of events protein
synthesis is similar for both eukaryotic
and pro prokaryotic cells.
• In both cases the hydrolysis of GDP to
GDP is the source of energy for many of
the processes involved.
• However, the structures of prokaryotic
and eukaryotic ribosomes are different
prof. aza
• For example, the ribosomes of
prokaryotic cells of bacteria are
made up of 50S (see Apendix 3)
and 30S rRNA subunits whereas
the ribosomes of mammalian
eukaryotic cells consist ,of 60S and
40S rRNA subunits.
prof. aza
• The differences between the
ribosomes of prokaryotic and
eukaryotic ribosomes are the basis
of the selective action of some
antibiotics .
prof. aza
11.1. Prokaryotic CeLLs
• The first step in protein synthesis
is the correct alignment of mRNA
on the small subunit of the
ribosome.
• In prokaryotic cells this alignment
is believed to be due to binding by
base pairing between bases at the
3’ end of the rRNA of the ribosome
and bases at the 5’ end of the
mRNA.
prof. aza
• This ensures the correct alignment of
the AUG anticodon of the mRNA with
the P site of the ribosome.
• The mRNA sequence of bases
responsible for this binding occurs as
part of the upstream (5’ terminal end)
section of the strand before the start
codon.
prof. aza
• This sequence is often known as the
Shine-Dalgarno sequence after its
discovers. Shine-Dalgarno sequences
vary in length and base sequence (Figure
10.15).
• The initiating tRNA in prokaryotic cells
is a specific methionine-tRNA known as
tRNAfMet ,which is able to read the
start codon AUG but not when it is part
of the elongation sequence. tRNAfMet is
unique in that the methionine it carries
is usually in the form of its N-formyl
derivative.
prof. aza
Figure 10.15. Examples of Shine-Dalgarno
sequences (bold larger type) of mRNA recognised
by Escherichia coli ribosomes. These sequences
lie about 10 nucleotides upstream of the AUG
start codon for the specified protein.
prof. aza
When AUG is part of the
elongation sequence methionine is
added to the growing protein by a
different transfer RNA known as
tRNAmMet, which also has the
anticodon UAC.
prof. aza
• However, tRNAmMet cannot initiate
protein synthesis. Elongation follows the
general mechanism for protein synthesis
(see section 10.9).
• It requires a group of proteins known as
elongation factors and energy supplied
by the hydrolysis of GTP to GDP.
Termination normally involves three
release factors.
prof. aza
• Experimental work has shown that
an mRNA strand actively
synthesizing proteins still have
several ribosomes attached to it at
different places along its length.
These multiple ribosome structures
are referred to as polyribosomes or
polysomes.
prof. aza
• The polysomes of prokaryotic cells
can contain up to 10 ribosomes at
any one lime. Each of these
ribosomes will be simultaneously
producing the same polypeptide or
protein;
prof. aza
• the further the ribosome has
moved along the mRNA, the longer
the polypeptide chain. The process
resembles the assembly line in a
factory. Each mRNA strand can in
its lifetime produce up to 300
protein molecules.
prof. aza
10 amino acid residues are added..
• In prokaryotic but not eukaryotic cells
(see section 4.1), ribosomes are found in
association with DNA.
• This is believed to he due to the
ribosome binding to the mRNA as it is
produced by transcription from the
DNA.
prof. aza
• Furthermore, these ribosomes have
been shown to start producing the
polypeptide chain of their
designated protein before
transcription is complete.
prof. aza
• This means that in bacteria protein
synthesis can be very rapid and in
some cases faster than
transcription. It has been reported
that in some bacteria an average of
10 amino acid residues are added to
the peptide chain ever second
prof. aza
11.2. Eukaryotic Cells
• The initiation of protein synthesis
in eukaryotic cells follows a
different route from that found in
prokaryotic cells although it still
uses a methionine-tRNA to start
the process.
prof. aza
• Eukaryotic mRNAs has no Shine-
Dalgarno sequences but are
characterised by a 7-methyl GTP
unit at the 5’ end of the mRNA
strand and a polyadenosine
nucleotide tail at the 3’ end of the
strand (Figure 10.16).
prof. aza
prof. aza
• In eukaryotic cells, the initiating
tRNA is a unique form of the
activated methionine- tRNA (tRNAi
Met). However, unlike in the case of
prokaryotic cells, the methionine
residue it carries is not formylated.
prof. aza
• The initiating process is started by
this tRNAi Met binding to the 40S
subunit of the ribosome to form
the so-called preinitiation complex,
the process requiring the formation
of a complex between tRNAi Met ,
various eukaryotic initiation factors
(elFs) and GTP.
prof. aza
• At this point the mRNA binds to
the 40S preinitiation complex. This
binding process is believed to
involve a number of eukaryotic
initiation factors and energy
supplied by the conversions of GTP
to GDP and ATP to ADP. Once the
mRNA has bound to the
preinitiation complex the 60S
subunit recombines with the 40S
prof. aza
• Once the mRNA has bound to
the preinitiation complex the
60S subunit recombines with
the 40S unit to form the
initiation complex (Figure 10.17).
prof. aza
• The initiating process is started by
this tRNAi Met binding to the 40S
subunit of the ribosome to form
the so-called preinitiation complex,
the process requiring the formation
of a complex between tRNAi Met ,
various eukaryotic initiation factors
(elFs) and GTP.
prof. aza
• The absence of the Shine-Dalgarno
sequence means that an alternative
mechanism must he available to
align the first AUG codon of the
mRNA with the P site of the
ribosome. This mechanism is
believed to direct the preinitiation
complex to the first AUG codon of
the mRNA.
prof. aza
• Elongation in eukaryotic ribosomes
follows the general mechanism for
protein synthesis (see section 10.10.3)
but involves different factors and
proteins from those utilised by
prokaryotic ribosomes. Termination only
requires one release factor, unlike in
prokaryotic ribosomes-where three
release factors are usually required.
prof. aza
• Elongation in eukaryotic ribosomes
follows the general mechanism for
protein synthesis (see section 10.10.3)
but involves different factors and
proteins from those utilised by
prokaryotic ribosomes.
• Termination only requires one release
factor, unlike in prokaryotic ribosomeswhere three release factors are usually
required.
prof. aza
• Termination only requires one
release factor, unlike in
prokaryotic ribosomes-where
three release factors are
usually required.
prof. aza
Figure 10.17. An outline of the formation of the
protein synthesis initiation complex by the
ribosomes of eukaryotic cells.
prof. aza