A. Decoding According to Base Pairing
Comparison of the anticodon of the incoming AA–tRNA
with the corresponding mRNA codon takes place at the
decoding center of the ribosome, located on the small subunit.
Peptide bond formation occurs on the large ribosomal
subunit, at the peptidyl transferase center. Thus, this segregation
of functions parallels the two arms of the tRNA.
The anticodon portion of the tRNA binds to the small subunit,
where the genetic message is read. The acceptor arm
of tRNA (with its attached amino acid) contacts the large
subunit, where catalysis occurs.
Although the synthesis of a peptide bond is the key
step in translation, this is the easiest part of protein synthesis.
Once the amino group of an aminoacyl–tRNA is
properly positioned close enough to the carbonyl group
of a peptidyl–tRNA, peptide bond formation through nucleophilic
attack is energetically favorable. The ribosome
can be considered as a single enzyme whose function is
to catalyze peptide bond formation.
B. Elongation Factors
Addition of each incoming amino acid requires the cooperation
of three elongation factors. Elongation factor
Tu (EF-Tu) is the most abundant protein in E. coli,
about 100,000 copies per cell, or 5% of the cell’s protein.
This protein is a GTPase, and the EF-Tu:GTP complex
specifically binds aminoacyl–tRNAs (AA–tRNAs). Formation
of the ternary complex (EF-Tu:GTP:AA–tRNA)
protects the ester bond (linking the amino acid to its cognate
tRNA) from hydrolysis, and transports the AA–tRNA
to the ribosomal A-site. Once the correct codon–anticodon
interaction is confirmed, ribosome-triggered hydrolysis of
EF-Tu-bound GTP occurs. EF-Tu:GDP is then released
from the ribosome, and the AA–tRNA occupies the Asite.
While EF-Tu transports all elongator tRNAs aminoacylated
with natural amino acids to the ribosome, this
factor has negligible affinity for formylated or nonformylated
. The unpaired first position in the tRNAfMet
acceptor stem helix apparently is a negative recognition
element for EF-Tu:GTP, because this element prevents the
initiator tRNA from pairing with internal AUG or GUG
The elongation factor EF-Ts is a nucleotide exchange
factor that regenerates active EF-Tu:GTP (from EFTu:
GDP) for binding subsequent AA–tRNAs following
GTP hydrolysis. Before their functions were known, elongation
factors Tu and Ts were named for their observed
thermal stabilities in vitro
—Tu indicates that this protein
nstable, while Ts stands for T
table. In eukaryotes, the two subunits of elongation
factor EF-1 perform the functions of EF-Tu and EF-Ts.
Once the A-site is occupied by the incomingAA–tRNA,
nucleophilic attack on the peptidyl–tRNA by the AA–
tRNA occurs. The condensation reaction produces a new
peptide bond and lengthens the polypeptide chain by one
amino acid (Fig. 8). As a result, the growing protein is now
attached to the A-site tRNA; this addition to and transfer
of the polypeptide chain is called transpeptidation.
Following formation of the peptide bond, a major rearrangement
of components in the functional center of
the ribosome must take place. Because the most recently
entered tRNA has become the peptidyl–tRNA, it must
be moved from the A-site to the P-site. The former
peptidyl–tRNA has been deacylated and needs to vacate
the P-site. Finally the mRNA must move three nucleotides
further in the 3´-direction so that the next codon can be
read. The concerted movement of tRNAs and mRNA at
the end of each elongation round is called translocation,
and is catalyzed by elongation factor G (EF-G), another
of the GTPase proteins in the translational machinery.
|Figure 8 Steps in elongation. With the peptidyl–tRNA bound in the P-site and the incoming aminoacyl–tRNA in the
A-site, the peptidyl transferase activity of the large ribosomal subunit catalyzes peptide bond formation. The growing
polypeptide chain is then attached to the A-site-tRNA, and the deacylated tRNA is in the P-site. Elongation factor G
facilitates translocation of the peptidyl–tRNA to the P-site and the empty tRNA to the E-site prior to its release from
|Figure 9 Termination of protein synthesis and ribosome recycling.
In prokaryotes, RF1 hydrolyzes the newly synthesized protein
at stop codons UAG and UAA, while RF2 recognizes stop
codons UGA and UAA. The GTPase RF3 stimulates release of
either RF1 or RF2. In eukaryotes a single protein recognizes all
stop codons. The final step of translation is dissociation of the inactive
70S complex, stimulated by the ribosome recycling factor