Protein Biosynthesis Takes Place in Five Stages:
Step 1: Activation of Amino Acids
For the synthesis of a polypeptide with a defined sequence, two fundamental
chemical requirements must be met:
- The carboxyl group of each amino acid must be activated to facilitate formation
of a peptide bond
- A link must be established between each new amino acid and the information in
the mRNA that encodes it.
Both of these requirements are met by attaching the amino acid to a tRNA in the
first stage of protein synthesis. Attaching the right amino acid to the right tRNA
is critical. This reaction takes place in the cytosol, not the ribosome. When
attached to their amino acid (aminoacylated) the tRNAs are said to be ‘charged’.
Aminoacyl-tRNA Synthetases Attach the Correct Amino Acids to their
During the first stage of protein synthesis, taking place in the cytosol, aminoacyl-
tRNA synthetases esterify the 20 amino acids to their corresponding tRNAs. Each
enzyme is specific for one amino acid and one or more corresponding tRNAs.
Most organisms have one aminoacyl-tRNA synthetase for each amino acid. For
amino acids with two or more corresponding tRNAs, the same enzyme usually
aminoacylates all of them.
Aminoacylation of tRNA by aminoacyl-tRNA synthetases:
Step 1 is the formation of an aminoacyl adenylate, which remains bound to the
active site. In the second step, the aminoacyl group is transferred to the tRNA.
The mechanism of this step is somewhat different for the two classes of
For class I enzymes, the aminoacyl group is transferred initially to the 2’-
hydroxyl group of the 3’- terminal A residue, then to the 3’- hydroxyl group by a
transesterification reaction. For class II enzymes, the aminoacyl group is
transferred directly to the 3’- hydroxyl group of the terminal adenylate.
Proofreading by Aminoacyl-tRNA Synthetases:
The aminoacylation of tRNA accomplishes two ends:
- It activates an amino acid for peptide bond formation
It ensures appropriate placement of the amino acid in a growing polypeptide.
The identity of the amino acid attached to a tRNA is not checked on the ribosome,
so attachment of the correct amino acid to the tRNA is essential to the fidelity of
protein synthesis. Interaction between an Aminoacyl-tRNA Synthetase and a tRNA:
An individual Aminoacyl-tRNA synthetase must be specific not only for a single
amino acid but for certain tRNAs as well. The interaction between Aminoacyl-
tRNA synthetase and tRNA has been referred to as the ‘second genetic code’,
reflecting its critical role in maintaining the accuracy of protein synthesis.
Step 2: Initiation
The mRNA bearing the code for the polypeptide to be synthesized binds to the
smaller of two ribosomal subunits and to the initiating aminoacyl-tRNA. The
large ribosomal subunit then binds to form an initiation complex. The initiating
aminocyl-tRNA base-pairs with the mRNA codon AUG that signals the beginning
of the polypeptide. This process, which requires GTP, is promoted by cytosolic
proteins called initiation factors.
A Specific Amino Acid Initiates Protein Synthesis:
Protein synthesis begins at the amino-terminal end and proceeds by the
stepwise addition of amino acids to the carboxyl-terminal end of the growing
polypeptide. The AUG initiation codon thus specifies an amino-terminal
methionine residue. Although methionine has only one codon, (5’) AUG, all
organisms have 2 tRNAs for methionine. One is used exclusively whn (5’) AUG is
the initiation codon for protein synthesis. The other is used to code for a Met
residue in an internal position in a polypeptide.
The Three Steps of Initiation:
In step 1, the 30S ribosomal subunit binds two initiation factors, IF-1 and IF-3.
Factor IF-3 prevents the 30S and 50S subunits from combining prematurely. The
mRNA then binds to the 30S subunit. The initiating (5’) AUG is guided to its
correct position in the mRNA.
Bacterial ribosomes have 3 sites that bind tRNAs:
- The aminoacyl (A) site
- The Peptidyl (P) site
- The exit (E) site
The A and P sites bind to aminoacyl-tRNAs, whereas the E site blinds only to
uncharged tRNAS that have completed their task on the ribosome. Both the 30S
and 50S subunits contribute to the characteristics of the A and P sites, whereas
the E site is largely confined to the 50 subunit. The initiating (5’) AUG is
positioned at the P site, the only site to which fMet-tRNA fMetcan bind.
The fMet-tRNA fMeis the only aminoacyl-tRNA that binds to the P site; during the
subsequent elongation stage, all other incoming aminoacyl-tRNAs (including the
fMet-tRNA Metthat binds to interior AUG codons) bind first to the A site and only
subsequently to the P and E sites. The E site is the site from which the
‘uncharged’ tRNAs leave during elongation. Factor IF-1 binds at the A site and
prevents tRNA binding at this site during initiation.
In step 2 of the initiation process, the complex consisting of 30S ribosomal
subunit, IF-3, and mRNA is joined by both GTP-bound IF-2 and the initiating fMet-tRNA fMe. The anticodon of this tRNA now pairs correctly with the mRNA’s
In step 3, this large complex combines with the 50S ribosomal subunit;
simultaneously, the GTP bound to IF-2 is hydrolysed to GDP and Pi, which are
released from the complex. All three initiation factors depart from the ribosome
at this point.
Completion of these 3 steps produces a functional 70S ribosome called the
initiation complex, containing the mRNA and the initiating fMet-tRNA fMe. The
correct binding of fMet-tRNA to the P site in the complete 70S initiation
complex is assured by at least 3 points of recognition and attachment: the codon-
anticodon interaction involving the initiation AUG fixed in the P site, interaction
between the Shine-Dalgarno sequence in the mRNA and the 16S rRNA, and
binding interactions between the ribosomal P site and the fMet-tRNA fMetThe
initiation complex is now ready for elongation.
Step 3: Elongation
The nascent polypeptide is lengthened by covalent attachment of successive
amino acid units, each carried to the ribosome and correctly positioned by its
tRNA, which base