Download Translation is simply the decoding of nucleotide sequences on

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Translation is simply the decoding of nucleotide sequences on mRNA into the
amino acid sequence of a polypeptide. The process of Translation ( synthesis of a
polypeptide chain) can be divided into three rather distinct activities: a) Initiationwhich involves binding of ribosomal subunits with mRNA accompanied with tRNA
charged with methionine; b) Elongation- in which
molecules
successive aminoacylated tRNA
are brought sequentially on the ribosomes, i.e translating the mRNA
molecule and the formation of peptide bond between the successive amino acids, and
c) Termination-where the ribosomal units dissociate into individual units and the
synthesized polypeptide chain is released.
Proteins are the work horses of the cell, controlling virtually every reaction
within, as well as providing structure, and serving as signals to other cells. They are
long chains of amino acids, and the exact sequence of the amino acids determines the
final structure and function of the protein. Instructions for that sequence are encoded
in genes (DNA).The synthesis of every protein molecule in a cell is directed by an
mRNA, originally copied from DNA. Synthesis of RNA from DNA template is called
transcription and the process is catalyzed by an enzyme called RNA polymerase. Next
step is the synthesis of polypeptide (protein) from mRNA. Synthesis of a polypeptide
from mRNA includes two types of processes: (1) information-transfer process in which
the mRNA base sequence determines an amino acid sequence, and (2) chemical
process in which the amino acids are linked together. The complete series of events is
called Translation. Translation may also be defined as the process by which
amino
acid sequence of polypeptide is synthesized on a ribosomal complex according to the
nucleotide sequence of an mRNA molecule (Fig. 1).
Fig.1. Flow of genetic information from DNA to proteins
Before discussing the process of Translation, let us describe some
ingredients necessary for Translation. The main ingredients of Translation are
as under:
Messenger RNA: Messenger RNA (mRNA) is an RNA molecule that serves as a
template for protein synthesis. It is needed to bring the ribosomal subunits together
and to provide the coding sequence of bases that determines the amino acid sequence
in the resulting polypeptide chain.
Ribosomes: Ribosomes are the sites of protein synthesis ( Translation) in both
prokaryotes
and eukaryotes. They move along an
mRNA molecule and expose
codons for appropriate aminoacyl tRNA molecules as per genetic code (Fig. 2b). The
amino acids are added to the growing polypeptide chain one by one and the peptide
bond formation is catalyzed by rRNA of small subunit of ribosome. In Prokaryotes,
Translation occurs on 70S ribosomes whereas in Eukaryotes it occurs on 80S ribosomes
(except in mitochondria and chloroplasts)
The general structure of prokaryotic and eukaryotic ribosomes is similar, although
they differ in size (Fig. 2a). The small subunit (designated as 30S) of
prokaryotes
consists of the 16S rRNA and 21 proteins; the large subunit (50S) is composed of the
23S and 5S rRNA and 34 proteins. The subunits of
eukaryotic ribosomes are larger
and contain more proteins than their Eukaryotic counterparts have. The small subunit
(40S) of Eukaryotic ribosomes is composed of 18s rRNA and approximately 30
proteins; the larger subunit (60S) contain the 28S, 5.8S and 5S rRNA and about 45
proteins.
Transfer RNA ( tRNA): The sequence of amino acids in a polypeptide is determined
by
the
70S
80S
Fig. 2. a-­‐Structure of prokaryotic (70S) and eukaryotic (80S) ribosomes; b-­‐Genetic code showing t
aminoacids base sequence in the mRNA by means of a set of adaptor molecules known as tRNA .
The tRNA binds to the mRNA codons (group of three adjacent bases on mRNA) through
anticodon site, which contains three complementary bases to codons, and thus bind
amino acids adjacent to one another for the formation of peptide bond.
The tRNA’s are approximately 70-80 nucleotides long, and have characteristic
cloverleaf-like structure (Fig. 3a) that results from complementary base-pairing
between different regions of the molecule. All the tRNA molecules have sequence CCA
at their 3' terminus (amino acids are covalently attached to the ribose of the terminal A
of CCA), the anticodon loop at the other end, one DHU loop,
a pseudouridine loop
(PψC), and an extra arm or variable loop of 7-10 nucleotides.
X ray diffraction
analysis has shown that the tRNA’s are made up of two double helices arranged in the
shape of an ‘L’ (Fig. 3b).
Aminoacids: The pool of 20 amino acids is used in the synthesis of polypeptides or
proteins; these are shown in Fig. 2b.
Aminoacyl tRNA synthetases: This set of enzymes catalyses the attachment of each
amino acid to its corresponding tRNA molecule. A tRNA molecule attached to its amino
acid is called an aminoacylated tRNA or charged tRNA. The formation of charged tRNA
occurs in two steps:
I. Activation of amino acids: This reaction is brought about by binding of an amino
acid with the ATP, and is mediated by specific amino acid tRNAsynthetases, as a result
of this a complex, known as aminoacyl-AMP enzyme complex, is formed.
Amino acid + ATP
Aminoacid-AMP enzyme complex +PPi
II. Charging of an amino acid on tRNA: The aminoacid-AMP enzyme complex reacts
with a tRNA and transfers the amino acid to tRNA, accompanied with the release of
AMP and the enzyme. The reaction is highly specific.
Aminoacid-AMP enzyme complex + tRNA
Enzyme.
Charged tRNA + AMP +
a
b
Fig. 3. a-­‐ Two dimensional structure of tRNA; b-­‐ Comparison of two and three dim
of tRNA
The synthesis of a polypeptide, i,e. translation is a continues process; but for
the sake of simplicity, it is divided into three distinct steps, namely 1. Initiation,
2.
Elongation, and 3. Termination.
1. Initiation:
The main feature of the initiation is the binding of an mRNA to the
small subunit of ribosome, tRNA charged with formylated metionine, followed by the
large ribosomal subunit to form the initiation complex. The ribosomes have three sites
for tRNA molecules; these are:
E (exit) site, from which the uncharged tRNA leave
during elongation, P (peptidyl) site, and an A (aminoacyl) site. The initiation proceeds
with the binding of the 30S ribosomal subunit with the initiation factors, IF-1 & IF-3.
The IF-3 prevents the 30S & 50S sub units from combining prematurely. Factor IF-1
binds at the A site during initiation. The mRNA then binds to the 30S subunit in such a
way that the initiating (5')AUG codon is positioned at the P site of ribosome. Bacterial
mRNA posses a specific sequence of nucleotides (called the Shine-Dalgarno sequence,
named after its discoverers) that resides 5-10 nucleotides before the initiation codon.
The Shine-Dalgarno sequence is complementary to a sequence of nucleotides near the
3' end of the 16s rRNA of small ribosomal subunit. It is pairing
between
complementary bases of Shine-Dalgarno sequence and 16S rRNA, that positions AUG
codon at P site of ribosome. The initiation complex consisting of the 30S ribosomal
subunit , IF-3, IF-1, and mRNA is joined by both GTP bound IF-2 and the initiating
tRNA-fmet. The anticodon of this
tRNA
pairs with the initiation codon of mRNA. It
should be noted that only the first charged tRNA binds with the P site, all the other
successively added tRNA attaches at A site of ribosomes. This is followed by joining of
50S ribosomal subunit, but at this step GTP bound to IF-2 is hydrolyzed to GDP and
PPi, which are released from the complex. All the three initiation factors depart from
the ribosome at this point. At the completion of this process, a functional 70s
ribosome, called the initiation complex is formed (Fig. 4).
Initiation in eukaryotes proceeds in the similar way as in prokaryotes, i,e.
binding of small subunit (40S) with the mRNA followed by charged tRNA and then
larger subunit (60S) to form the initiation complex; but initiation in eukaryotes requires
atleast 10 initiation factors, designated as eIFs ( Eukaryotic initiation factors), which
makes initiation in eukaryotes more complex.
Fig. 4. Formation of initiation complex at the expense of hydrolysis of ATP
2. Elongation:
After the initiation complex has been formed, translation proceeds
by elongation of the polypeptide chain. The ribosome has three sites for tRNA binding,
designated the P site (Peptidyl), A site ( Aminoacyl), and E (Exit) site as mentioned
earlier. The initiator methionyl tRNA is bound at the P site.
The first step in elongation is binding of the next aminoacyl tRNA to the A site
by pairing with the second codon of the mRNA. The aminoacyl tRNA is escorted to the
A site of the ribosome by an elongation factor EF-TU in prokaryotes, which is
complexed with GTP. The GTP is hydrolyzed to GDP as the correct aminoacyl tRNA is
inserted into the A site of the ribosome, and the elongation factor bound to GDP is
released. The hydrolysis of GTP in elongation is the rate-limiting step, and provides the
time interval during which an incorrect aminoacyl tRNA (which would bind less strongly
to the mRNA codon) can dissociate from the ribosome, rather than being used in the
formation of a polypeptide. The expenditure of a high energy GTP at this step has an
important contribution to accurate protein synthesis. It allows time for proof reading of
the codon-anticodon pairing before the peptide bond forms.
Fig. 5. Eongation (the binding of the second aminoacyl-tRNA and formation of a
polypeptide bond)
Once EF-TU has left the ribosome, a peptide bond is formed between the amino
acids attached with the tRNA on A site and P site (Fig. 5.1). This reaction is catalyzed
by the large ribosomal subunit, with
RNA playing a critical role; the result is the
transfer of the first amino acid at P site to the aminoacyl tRNA at the A site of the
ribosome, forming a dipeptide tRNA at this position, and leaving the uncharged initiator
tRNA at the P site. The next step in elongation is translocation, which requires another
elongation factor known as translocase (or EF-G in prokaryotes), and is again coupled
to GTP hydrolysis. During translocation, the ribosome’s move three nucleotides along
the mRNA in 5' → 3' direction at each step, positioning the next codon in an empty A
site. The binding of a new aminoacyl tRNA to the A site then induces the release of the
uncharged (deacylated) tRNA from the E site into the cytosol (Fig. 5.2). At each step of
translocation, newer and newer aminoacyl tRNA attaches at the A site, and uncharged
tRNA are exited through the E site, as the ribosome moves from codon to codon along
the mRNA towards the 3' end.
As elongation continues, the EF-TU that is released from the ribosome bound to
GDP, must be reconverted to its GTP form. This conversion requires a third elongation
factor, EF-Ts, which binds to EF-TU/GDP complex, and promotes the exchange of
bound GDP for GTP. This exchange results in the regeneration of EF-TU/GTP, which is
now ready to escort a new aminoacyl tRNA in the A site of the ribosome, beginning a
new cycle of elongation.
Fig 5.2 Elongation (the ribosomes moves one codon towords the 3' end of mRNA translocation)
The elongation cycle in eukaryotes is quite similar to that of prokaryotes. The three
elongation factors eEF-1α, eEF-1βγ and eEF-2 in eukaryotes are analogous to that of
prokaryotic
elongation
factors
EF-TU,
EF-TS
and
EF-G,
respectively.
Further
eukaryotic ribosomes do not have E site, so uncharged tRNA are expelled directly from
the P site.
3. Termination: Elongation
polypeptide
codon,
continues
also
(UAA,
called
UAG
until
a
nonsense
or
UGA),
of
a
stop
codon
is
translocated into the A site of
the
ribosome . Cells do not contain
tRNA
with anticodon complimentary to
these
terminating signals ,
have
release
recognize
instead
factors
that
signals
and
the
they
terminate protein synthesis (Fig
The
release
factors
6).
have
domains thought to mimic the
structure of tRNA. Prokaryotic
cells
contain two release factors that
recognize
codon;
RF-1
recognizes UAA or UAG, and RF-
2
recognizes
termination
UAA
or
UGA.
In
eukaryotes, a single releasing
factor
eRF-1 recognizes all the three
termination codons. The release
factors bind to the termination
at
the
A
hydrolysis
site
of
and
stimulate
bond
between
and polypeptide chain at the P
resulting
in
the
release
codon
tRNA
site
,
of
polypeptide from the ribosome.
Both
prokaryotic and eukaryotic cells
also
Fig. 6 The termination of protein synthesis in response to a termination codon. contain release factors, RF-3 and
eRF-3, respectively, that don’t recognize specific
termination codon, but act together with RF-1 (eRF-1), and RF-2 and are thought to
dissociate or release the ribosomal units.
In most of the cells, single mRNA can be translated simultaneously by several
ribosomes in both prokaryotic and eukaryotic ones (particularly in metabolically active
cells). As one ribosome
moves from the initiation site, another one can bind to the
mRNA and begins synthesis of a new polypeptide chain, such a condition is known as
polysome or polyribosome. This type of translation helps the cell to amplify the specific
protein and meet the excessive demand of the protein within or outside the cell.
Fig. 7. Polysome (where more than one ribosome translate same mRNA simultaneously)