Process of Translation

Translation can be categorized into

Initiation

Elongation

Termination

Initiation involves binding of a ribosome to mRNA. Elongation involves repeated addition of

amino acids and termination involves release of the new polypeptide chain. Sets of accessory
proteins assist the ribosomes in each of the three stages. Translation requires the use of
energy by the cell which is provided by hydrolysis of GTP and ATP. GTP is used for
ribosome movement and in binding of accessory factors. ATP is used to charge tRNAs and in
removing secondary structure from mRNAs. Upto 90% of ATP produced in a bacterium is
used for translation. One peptide bond formation during prokaryotic protein synthesis utilizes
two GTP and one ATP molecules.

4.1 Initiation: The process of initiation in prokaryotes is much different from that in
eukaryotes. This is due to the dissimilar nature of mRNA in both prokaryotes and eukaryotes.

Binding of Small Ribosomal Subunit to mRNA-Ribosomes are present as separate large and
small subunits when they are not engaged in translation. The binding of the small subunit of
the ribosome to the mRNA marks the first step of translation process. Translation starts at the
sequence AUG. Upstream of AUG, the small subunit of ribosome binds at a particular point
to mRNA. In prokaryotes, this is known as the Shine-Dalgarno sequence
(5’AGGAGGU3’)that is found near the start of mRNA. After binding to sequence, the small
subunit of ribosome now drifts in a 3’ direction along tit hits upon AUG, about 10
nucleotides downstream.
Formation of Initiation Complex-In bacteria, the first amino acid on the mRNA is different as
it has an addition of a formyl (-CHO) group to one of the hydrogens of the amino group. This
obstructs the amino group, thus preventing it from forming a peptide bond. In this way it
ensures that polymerization of the polypeptide can only occur in the amino to carboxy
direction. The mRNA, the small ribosomal subunit and tRNAfmet together forms the
Initiation Complex. Two types of tRNAs recognize AUG and carry methionine. One is used
for initiation (tRNAfmet) and the other recognizes internal AUGs. Only the initiator tRNA is
capable of binding to the initiation complex (Fig.6). Since N-formyl methionine is the
initiator amino acid, so it can be thought that almost all prokaryotic proteins will have formyl
group in the beginning. This is not true as there is an

enzyme “deformylase” that cleaves this for synthesized protein in prokaryotes.

Role of Initiation Factors- Certain accessory proteins calledalso “ required for initiation. IF1,
IF2 and IF3 are the three initiation factors in bacteria (Fig.7).

Each factor has a specific role to play in the initiation process.

The process of Initiation starts with the binding of IF1 and IF3 to the small ribosomal
subunit. IF1 ensures that tRNA does not get bound to A site of the small ribosomal subunit.

Role of IF3 is to ensure that the large subunit does not bind prematurely before mRNA has
bound. In fact IF3 gets bound to the small ribosomal subunit in the previous cycle of
translation itself and hence ensures dissociation of ribosome into its large and small subunits.

IF2 is actually a GTPase. It gets bound to GTP and together they bind to the small subunit.
This assists in binding of the initiator tRNA. Now the small subunit binds to the mRNA and
finds the AUG codon where the translation has to start. The initiator tRNA carrying
methionine binds to the complex and IF3 is released. This

symbolizes the end of initiation.

Elongation: Unlike initiation, the process of translation is highly conserved in both

prokaryotes and eukaryotes. Once the ribosome assembly has taken place along with the
initiator tRNA at P site, the protein synthesis commences as follows-
Binding of incoming aminoacyl tRNA at A site- The next step involves the release of IF1 and
IF2 and GTP hydrolysis takes place. The complete ribosome now has two binding sites for
tRNA molecules. The tRNAfmet base paired to AUG occupies the Peptidyl site or the P-site.
The second site is the A site or aminoacyl site and is located over the second codon.
Elongation commences when a tRNA enters the A site. This tRNA now base pairs with the
second codon (Fig.8).

Formation of Peptide Bond- Once both the sites in the ribosome are occupied by charged
tRNAs, the attached amino acids are placed in close contact and peptide bond can form
between the carboxyl group of the methionine and amino group of the second amino acid.
The reaction is catalyzed by an enzyme peptidyl transferase. Peptidyl transferase works in
concurrence with another enzyme, tRNA deacylase, which breaks the link between
methioinine and its tRNA after formation of the peptide bond.

Certain elongation factors or accessory proteins are also required for elongation. In
prokaryotes, two elongation factors EFTu and EFTs are involved (Fig. 9).

EFTu is involved with entry of a tRNA in to the A site. EFTu binds charged tRNAs in
association with GTP. Following entry to the A site, the GTP is hydrolyzed and EFTu is
released bound to GDP. In this state, EFTu has least affinity for aminoacyl tRNA. On its own
EFTu associated with aminoacyl tRNA does not have any GTPase activity. This actually gets
activated when EFTu gets associated with the same domain on large ribosomal subunit
(factor binding center) that also activates IF2 GTPase. Again this can take place only if the
correct codon-anticodon binding has taken place. Hence this becomes a very important
regulatory step of prokaryotic protein synthesis

Before another tRNA can bind, EFTu must be regenerated with the help of EFTs. First EFTs
displaces GDP by binding to EFTu, a new molecule of GTP then combines to EFTu and
releases EFTs.

Translocation-

Following peptide bond formation, translocation occurs and the ribosome shifts to the
following codon. The newly formed protein chain with two amino acids is bound to the
second tRNA. It now moves into the P site expelling the uncharged tRNA and the A site
becomes free or unoccupied. A third charged tRNA enters the A site and the elongation cycle
is replicated (Fig. 8). Following each addition of an amino acid to the growing polypeptide
chain, the ribosome translocates to the next codon. In bacteria, translocation is mediated by
the elongation factor EFG which gets bound to the ribosome in a complex with GTP. This is
then hydrolyzed to provide energy for translocation. Binding of EFG and EF-Tu to the
ribosome is mutually exclusive. This ensures that translocation is completed before the next
round of elongation begins.

As translocation proceeds, the ribosomes moves along the mRNA away from the initiation
site which is then free to bind a new ribosome. Several ribosomes can bind to a single mRNA
during the process of protein synthesis, forming a structure known as Polysome /
Polyribosome

Proofreading mechanism of Ribosomes: Ribosomes make use of three mechanisms to ensure

that incorporation of incorrect aminoacyl tRNA is avoided. The rate of error is almost 10-4.

The presence of adjacent A-A residues on 16srRNA at A site of small subunit forms
hydrogen bonding with the minor groove of codon-anticodon pair. This takes place only if
there is correct base pairing between codon and anticodon.

Secondly, the correct pairing also ensures efficient GTPase activity associated with release of
EFTu. In the absence of correct pairing EFTu is not at its proper place and its associated
GTPase activity is hindered.

Thirdly, correctly base paired aminoacyl tRNA remain associated with ribosome and this

4.3 Termination: When a termination codon enters the A site, it marks the end of translation.
The completed polypeptide is now released. There are no tRNAs that can bind to the
termination codons. Instead proteins called release factors enter the A site and causes
the release ofcompleted polypeptide. In E.coli, two release factors RF1 and RF2 perform
this function. RF1identifies UAA, UAG while RF2 recognizes UAA, UGA. Another factor
RF3 plays an ancillary role. The release factor also promotes the transfer of the polypeptide
to a water molecule by peptidyl transferase rather than to another aminoacyl tRNA (Fig. 11).
To complete the ribosomecycle, a class II release factor, a ribosome recycling factor and IF3
(initiation factor) ensures complete termination of translation followed by dissociation of
ribosome into large and small subunits that are ready to start another cycle.

Summary-
Translation in Prokaryotes is a process that involves decoding the information of mRNA into
a chain of amino acids that finally forms the protein. Four principal components are involved
in the process of protein synthesis. They are: mRNA, tRNA, aminoacyl tRNA synthetase and
the ribosomes. The entire process can be categorized into three major steps-Initiation,
Elongation and Termination. Initiation results in formation of initiation complex wherein
small unit of ribosome is bound to mRNA at the start site and the initiator tRNA is bound to
the first or initiator codon, bound to ribosome. Elongation leads to joining of amino acids to
form a polypeptide based on the information given by the mRNA molecule. The process of
elongation comes to an end once the termination codon is reached on mRNA. This marks the
end of elongation and beginning of

Termination that leads to release of ready-made protein from the ribosome. This also marks
the end of ribosome cycle. Newly synthesized protein may undergo post translational
modifications to be fully functional. Certain accessory factors like IF1, IF2 and IF3 in
Initiation; EFTu, EFTs and EFG in Elongation and RF1, RF2 and RF3 play important role
during this process. The process of translation ensures the consistency of mRNA, as the ones
that are incomplete (e.g. devoid of stop codons) may produce truncated proteins that are
degraded by cellular proteases.