Protein Synthesis

The events in protein synthesis are better known in bacteria than in eukaryotes.
Although these are thought to be similar in the two groups, some differences do occur.
The following description refers mainly to protein synthesis in bacteria on the 70S
ribosomes.
It involves 4 steps: -
1. Activation of Amino Acids:
Amino acid activation also known as aminoacylation or tRNA charging refers to the
attachment of an amino acid to its Transfer RNA (tRNA).
During amino acid activation the amino acids are attached to their corresponding
tRNA. The reactions are catalysed by a group of enzymes called aminoacyl-tRNA
synthetases. The reaction proceeds in 2 steps –
aa + ATP → aa.AMP + PP (Pyrophosphate)
aa.AMP + tRNA → aa.tRNA +AMP
The amino acid is covalently bonded via ester bond at the adenosine 3’ end of the
tRNA at the expense of an ATP. The formation of ester bond conserves a considerable
part of energy from the activation reaction. This stored energy provides the majority
of energy needed for peptide bond formation during translation.
Each of the 20 amino acids are recognized by its specific aminoacyl-tRNA synthetase.
In Prokaryotes, the starting amino acid at the amino terminal end is always N-formyl
methionine. N-formyl methionine is attached by N-formyl methionyl-tRNA synthetase.
N-formyl methionine is attached to tRNAfmet and forms N-formylmethionyl tRNA.
There are 2 species of tRNA specific for methionine, tRNAfmet and tRNAmet. tRNAmet
accepts methionine and is used to insert methionine in interior position in polypeptide
chain.
In Eukaryotes, a special class of tRNAs called initiator tRNAs (tRNAi) carry methionine
or its derivative, formyl-methionine to ribosome to start a highly regulated process of
protein synthesis.
2. Initiation of Polypeptide Synthesis:
The conditions required for initiation are
i) m-RNA with initiation codon AUG ii) N-formyl methionyl- t-RNA iii) GTP
iv) 30S and 50S subunits of ribosome v) Mg2+ vi) Initiation Factors IF1, IF2, and IF3
Initiation of translation involves 4 steps.
a) Formation of IF3-IF1-30S complex:
Ribosome exists as 70S subunit in the presence of Mg2+ ions. When the Mg2+
concentration decreased, 70S subunit dissociated into 50S and 30S subunit. The
initiation factor IF3 and IF-1 binds with the 30S subunit so as to bring about change in
shape of 30S subunit which prevents its association with 50S subunit.
b) Binding of m-RNA:
The initiating codon in m-RNA binds to the m-RNA binding site located on 30S subunit.
The initiation codon AUG is guided to correct position on 30S subunit by a special signal
sequence called Shine-Dalgarno sequence (AGGAGG). This stage results in the
formation of IF3-IF1-30S-m-RNA complex.
c) Binding of N-formylmethionyl- t-RNA to IF3-IF1-30S-m-RNA complex:
Since there is only one codon for methionine which codes for both initiating and
interior methionine residues, the initiation signal of Shine-Dalgarno sequence identify
the site where N-formylmethionyl-t-RNA is to be bound. Interior AUG codons are
specific for methionyl t-RNA and cannot bind N-formylmethionyl t-RNA. IF-2 which
already contains GTP and N-formylmethionyl t-RNA binds to IF-3-IF-1-30Sm-RNA
complex During the binding, the t-RNA is correctly placed on initiating codon.
d) Formation of Initiation Complex:
In this stage, 50S ribosomal subunit combines with large complex with the
simultaneous release of initiation factors IF-1, IF-2 and IF-3 which is accompanied by
the hydrolysis of GTP to GDP and Pi. This give rise to a functional initiation complex
containing N-formylmethionyl tRNA –m-RNA -70S ribosome.
3. Elongation:
It occurs in three steps namely;
a) Binding of aminoacyl t-RNA to A – Site:
At this point N-formylmethionyl- tRNA molecule in the 70S initiation complex occupies
the P site on the ribosome. The other site for a tRNA molecule, i.e. the A site, is empty.
The N-formylmethionyl-tRNA is positioned in such a way that its anticodon pairs with
the initiating AUG (or GUG) codon on mRNA. (Alternative start codons different from standard
AUG codon are found in both prokaryotes and eukaryotes. Alternate start codons are still translated
as Met when they are at the start of a protein (even if the codon encodes a different amino acid
otherwise). This is because a separate transfer RNA (tRNA) is used for initiation.)

The elongation cycle in the protein synthesis begins with the insertion of an aminoacyl
tRNA into the empty A site on the ribosome. The species of tRNA to be inserted
depends upon the mRNA codon that is present in the A site. The complementary
aminoacyl tRNA is transferred to the A site by a non-ribosomal specific cytoplasmic
protein, called the elongation factor T (EF-T) that binds to the aminoacyl tRNA. The
factor EF-T contains two subunits, EF-Ts and EF-Tu.
First aminoacyl t-RNA binds with a binary complex of EF-TU-GTP to form a ternary
(composed of three parts) complex.
It binds to ribosome and during this reaction GTP hydrolyzed into GDP and Pi. After
binding, EF-TU-GDP and Pi released. EF-TU-GDP then binds with EF-TS to form EF-TS-
EF-TU-GDP complex. GTP then replaces GDP to form EF-TU-GTP along with the release
of EF-TS. This EF-TU-GTP complex now ready for the addition of another aminoacyl
tRNA to A site.
b) Peptide bond formation:
Once the initiator N-formylmethionyl-tRNA occupies the P site and the next aminoacyl-
tRNA occupies the A site, a peptide bond between the adjacent amino acids is formed
by an enzyme, peptidyl transferase belonging to the 50S subunit. The active site of the
peptidyl transferase is the 23 S rRNA. The uncharged tRNAfmet occupies the P site and
the dipeptide formed is attached to the second tRNA occupying the A site following
the formation of a peptide bond. The product of the first peptide bond formation is
called dipeptidyl-tRNA bound to the A site.
c) Translocation:
In the third step of elongation cycle, the ribosomes move along m-RNA towards its 3’-
end by the distance of one codon. This shift of ribosome along m-RNA is called
translocation. This requires elongation factor EF-G which is also called as Translocase.
The hydrolysis of GTP provides energy for Translocation.
4. Termination:
Two conditions are necessary for termination of protein synthesis. One is the presence
of a stop codon that signals the chain elongation to terminate, and the other is the
presence of release factors (RF) which recognise the chain terminating signal. There
are three terminating codons, UAA, UGA and UAG for which tRNAs do not exist.
Behind all this complexity is the fact that after the polypeptide chain has reached its
full length, its carboxyl end is still bound to its tRNA adapter.
Termination must, therefore, involve the splitting of the terminal tRNA. Release of the
peptidyl tRNA from the ribosome is promoted by three specific release factors, RF1,
RF2 and RF3. RF1 recognises triplets UAA and UAG, while RF2 recognises UAA and
UGA. The third factor RF3 does not possess any release activity of its own, but it binds
to GTP and stimulates the binding of RF1 and RF2 with the ribosome.