Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

344 Chapter 6: How Cells Read the Genome: From DNA to Protein
Figure 6–65 Detailed view of the translation cycle. The outline of
translation presented in Figure 6–64 has been expanded to show the roles
of the two elongation factors EF-Tu and EF-G, which drive translation in the
forward direction. As explained in the text, EF-Tu provides opportunities for
proofreading of the codon –anticodon match. In this way, incorrectly paired
tRNAs are selectively rejected, and the accuracy of translation is improved.
The binding of a molecule of EF-G to the ribosome and the subsequent
hydrolysis of GTP lead to a rearrangement of the ribosome structure, moving
the mRNA being decoded exactly three nucleotides through it (Movie 6.9).
AP
A
5′ 3′
mRNA
E site
P site A site
GTP
EF-Tu
without the aid of these elongation factors and GTP hydrolysis, but this synthesis
is very slow, inefficient, and inaccurate. Coupling the GTP hydrolysis-driven
changes in the elongation factors to transitions between different states of the
ribosome speeds up protein synthesis enormously. The cycles of elongation factor
association, GTP hydrolysis, and dissociation also ensure that all such changes
occur in the “forward” direction, helping translation to proceed efficiently (Figure
6–65).
In addition to moving translation forward, EF-Tu increases its accuracy. As we
discussed in Chapter 3, EF-Tu can simultaneously bind GTP and aminoacyl-tR-
NAs (see Figures 3–72 and 3–73), and it is in this form that the initial codon–anticodon
interaction occurs in the A site of the ribosome. Because of the free-energy
change associated with base-pair formation, a correct codon–anticodon match
will bind more tightly than an incorrect interaction. However, this difference in
affinity is relatively modest and cannot by itself account for the high accuracy of
translation.
To increase the accuracy of this binding reaction, the ribosome and EF-Tu
work together in the following ways. First, the 16s rRNA in the small subunit of
the ribosome assesses the “correctness” of the codon–anticodon match by folding
around it and probing its molecular details (Figure 6–66). When a correct match
is found, the rRNA closes tightly around the codon–anticodon pair, causing a conformational
change in the ribosome that triggers GTP hydrolysis by EF-Tu. Only
when GTP is hydrolyzed does EF-Tu release its grip on the aminoacyl-tRNA and
allow it to be used in protein synthesis. Incorrect codon–anticodon matches do
not readily trigger this conformational change, and these errant tRNAs mostly fall
off the ribosome before they can be used in protein synthesis. Proofreading, however,
does not end here.
After GTP is hydrolyzed and EF-Tu dissociates from the ribosome, there is a
second opportunity for the ribosome to prevent an incorrect amino acid from
being added to the growing chain. There is a short time delay as the amino acid
carried by the tRNA moves into position on the ribosome. This time delay is
shorter for correct than incorrect codon–anticodon pairs. Moreover, incorrectly
matched tRNAs dissociate more rapidly than those correctly bound because their
interaction with the codon is weaker. Thus, most incorrectly bound tRNA molecules
(as well as a significant number of correctly bound molecules) will leave the
ribosome without being used for protein synthesis. The two proofreading steps,
acting in series, are largely responsible for the 99.99% accuracy of the ribosome in
translating RNA into protein.
Even if the wrong amino acid slips through the proofreading steps just
described and is incorporated onto the growing polypeptide chain, there is still
one more opportunity for the ribosome to detect the error and provide a solution,
albeit one that is not, strictly speaking, proofreading. An incorrect codon‒
anticodon interaction in the P site of the ribosome (which would occur after the
misincorporation) causes an increased rate of misreading in the A site. Successive
rounds of amino acid misincorporation eventually lead to premature termination
of the protein by release factors, which are described below. Normally,
these release factors act when translation of a protein is complete; here, they act
early. Although this mechanism does not correct the original error, it releases the
flawed protein for degradation, ensuring that no additional peptide synthesis is
wasted on it.
PROOFREADING
PROOFREADING
P
P
P
P
A
A
A
AE
P AP A
E
A
P
P i
A
GTP
incorrectly basepaired
tRNAs
preferentially
dissociate
P i
GDP
GDP
incorrectly basepaired
tRNAs
preferentially
dissociate
GTP
GTP
EF-G
GDP