Protein Factors Play Key Roles in Protein Synthesis

III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.4. Protein Factors Play Key Roles in Protein Synthesis
Although rRNA is paramount in the process of translation, protein factors also are required for the efficient synthesis of
a protein. Protein factors participate in the initiation, elongation, and termination of protein synthesis. P-loop NTPases of
the G-protein family play particularly important roles. Recall that these proteins serve as molecular switches as they
cycle between a GTP-bound form and a GDP-bound form (Section 15.1.2).
29.4.1. Formylmethionyl-tRNAf Is Placed in the P Site of the Ribosome During
Formation of the 70S Initiation Complex
Messenger RNA and formylmethionyl-tRNAf must be brought to the ribosome for protein synthesis to begin. How is
this accomplished? Three protein initiation factors (IF1, IF2, and IF3) are essential. The 30S ribosomal subunit first
forms a complex with IF1 and IF3 (Figure 29.27). The binding of these factors to the 30S subunit prevents it from
prematurely joining the 50S subunit to form a dead-end 70S complex, devoid of mRNA and fMet-tRNAf. Initiation
factor 2, a member of the G-protein family, binds GTP, and the concomitant conformational change enables IF2 to
associate with formylmethionyl-tRNAf. The IF2-GTP-initiator tRNA complex binds with mRNA (correctly positioned
by the Shine-Dalgarno sequence interaction with the 16S rRNA) and the 30S subunit to form the 30S initiation complex.
The hydrolysis of GTP bound to IF2 on entry of the 50S subunit leads to the release of the initiation factors. The result is
a 70S initiation complex.
When the 70S initiation complex has been formed, the ribosome is ready for the elongation phase of protein synthesis.
The fMet-tRNAf molecule occupies the P site on the ribosome. The other two sites for tRNA molecules, the A site and
the E site, are empty. Formylmethionyl-tRNAf is positioned so that its anticodon pairs with the initiating AUG (or GUG)
codon on mRNA. This interaction sets the reading frame for the translation of the entire mRNA.
29.4.2. Elongation Factors Deliver Aminoacyl-tRNA to the Ribosome
The second phase of protein synthesis is the elongation cycle. This phase begins with the insertion of an aminoacyltRNA into the empty A site on the ribosome. The particular species inserted depends on the mRNA codon in the A site.
The cognate aminoacyl-tRNA does not simply leave the synthetase and diffuse to the A site. Rather, it is delivered to the
A site in association with a 43-kd protein called elongation factor Tu (EF-Tu). Elongation factor Tu, another member of
the G-protein family, binds aminoacyl-tRNA only in the GTP form (Figure 29.28). The binding of EF-Tu to aminoacyltRNA serves two functions. First, EF-Tu protects the delicate ester linkage in aminoacyl-tRNA from hydrolysis. Second,
the GTP in EF-Tu is hydrolyzed to GDP when an appropriate complex between the EF-Tu-aminoacyl-tRNA complex
and the ribosome has formed. If the anticodon is not properly paired with the codon, hydrolysis does not take place and
the aminoacyl-tRNA is not transferred to the ribosome. This mechanism allows the free energy of GTP hydrolysis to
contribute to the fidelity of protein synthesis.
How is EF-Tu in the GDP form reset to bind another aminoacyl-tRNA? Elongation Factor Ts, a second elongation
factor, joins the EF-Tu complex and induces the dissociation of GDP. Finally, GTP binds to EF-Tu, and EF-Ts is
concomitantly released. It is noteworthy that EF-Tu does not interact with fMet-tRNA . Hence, this initiator tRNA is not
f
delivered to the A site. In contrast, Met-tRNAm, like all other aminoacyl-tRNAs, does bind to EF-Tu. These findings
account for the fact that internal AUG codons are not read by the initiator tRNA. Conversely, initiation factor 2
recognizes fMet-tRNAf but no other tRNA.
This GTP-GDP cycle of EF-Tu is reminiscent of those of the heterotrimeric G proteins in signal transduction (Section
15.1.2) and the Ras proteins in growth control (Section 15.4.2). This similarity is due to their evolutionary heritage,
inasmuch as the amino-terminal domain of EF-Tu is homologous to the P-loop NTPase domains in the other G proteins.
The other two domains of the tripartite EF-Tu are distinctive; they mediate interactions with aminoacyl-tRNA and the
ribosome. In all these related enzymes, the change in conformation between the GTP and the GDP forms leads to a
change in interaction partners. A further similarity is the requirement that an additional protein catalyze the exchange of
GTP for GDP; an activated receptor plays the role of EF-Ts for a heterotrimeric G protein, as does Sos for Ras.
29.4.3. The Formation of a Peptide Bond Is Followed by the GTP-Driven Translocation
of tRNAs and mRNA
After the correct aminoacyl-tRNA has been placed in the A site, the transfer of the polypeptide chain from the tRNA in
the P site is a spontaneous process, driven by the formation of the stronger peptide bond in place of the ester linkage.
However, protein synthesis cannot continue without the translocation of the mRNA and the tRNAs within the ribosome.
The mRNA must move by a distance of three nucleotides as the deacylated tRNA moves out of the P site into the E site
on the 30S subunit and the peptidyl-tRNA moves out of the A site into the P site on the 30S subunit. The result is that the
next codon is positioned in the A site for interaction with the incoming aminoacyl-tRNA.
Translocation is mediated by elongation factor G (EF-G, also called translocase). The structure of EF-G is exceptional
in revealing some aspects of its mode of action (Figure 29.29). The structure of EF-G closely resembles that of the
complex between EF-Tu and tRNA. This is an example of molecular mimicry; a protein domain evolved so that it
mimics the shape of a tRNA molecule. This structural similarity, as well as other experimental data, suggests a
mechanism for the translocation process (Figure 29.30). First, EF-G in the GTP form binds to the ribosome, primarily
through the interaction of its EF-Tu-like domain with the 50S subunit. The binding site includes proteins L11 and the L7L12 dimer. The tRNA-like domain of EF-G interacts with the 30S subunit. The binding of EF-G to the ribosome in this
manner stimulates the GTPase activity of EF-G. On GTP hydrolysis, EF-G undergoes a conformational change that
forces its arm deeper into the A site on the 30S subunit. To accommodate this domain, the peptidyl-tRNA in the A site
moves to the P site, carrying the mRNA and the deacylated tRNA with it. The ribosome may be prepared for these
rearrangements by the initial binding of EF-G as well. The dissociation of EF-G leaves the ribosome ready to accept the
next aminoacyl-tRNA into the A site.
29.4.4. Protein Synthesis Is Terminated by Release Factors That Read Stop Codons
The final phase of translation is termination. How does the synthesis of a polypeptide chain come to an end when a stop
codon is encountered? Aminoacyl-tRNA does not normally bind to the A site of a ribosome if the codon is UAA, UGA,
or UAG, because normal cells do not contain tRNAs with anticodons complementary to these stop signals. Instead, these
stop codons are recognized by release factors (RFs), which are proteins. One of these release factors, RF1, recognizes
UAA or UAG. A second factor, RF2, recognizes UAA or UGA. A third factor, RF3, another G protein homologous to
EF-Tu, mediates interactions between RF1 or RF2 and the ribosome.
Release factors use a Trojan horse strategy to free the polypeptide chain. One of the most impressive properties of the
ribosome is not that it catalyzes peptide-bond formation; the formation of a peptide bond by the reaction between an
amino group and an ester is a facile chemical reaction. Instead, a more impressive feature crucial to ribosome function is
that the peptidyl-tRNA ester linkage is not broken by premature hydrolysis. The exclusion of water from the peptidyl
transferase center is crucial in preventing such hydrolysis, which would lead to release of the polypeptide chain. The
structure of a prokaryotic release factor has not yet been determined. However, the structure of a eukaryotic release
factor, though probably not truly homologous to its prokaryotic counterpart, reveals the strategy (Figure 29.31).
The structure resembles that of a tRNA by molecular mimicry. The sequence Gly-Gly-Gln, present in both eukaryotes
and prokaryotes, occurs at the end of the structure corresponding to the acceptor stem of a tRNA. This region binds a
water molecule. Disguised as an aminoacyl-tRNA, the release factor may carry this water molecule into the peptidyl
transferase center and, assisted by the catalytic apparatus of the ribosome, promote this water molecule's attack on the
ester linkage, freeing the polypeptide chain. The detached polypeptide leaves the ribosome. Transfer RNA and
messenger RNA remain briefly attached to the 70S ribosome until the entire complex is dissociated in a GTP-dependent
fashion by ribosome release factor (RRF) and EF-G. Ribosome release factor is an essential factor for prokaryotic
translation.
The structure of RRF, too, resembles tRNA (Figure 29.32). However, the known tRNA-mimicking structures of
RRF, EF-G, and the release factors are distinct; they do not appear to have been generated from a common
ancestor. Thus, convergent evolution has provided a similar solution looking sufficiently like a tRNA to interact with
the tRNAbinding sites on the ribosome to several problems. The effects of divergentevolution are evident in the
protein factors that participate in translation, most notably in the form of the homologous G proteins, EF-Tu, EF-G, IF2,
and RF3.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.4. Protein Factors Play Key Roles in Protein Synthesis
Figure 29.27. Translation Initiation in Prokaryotes. Initiation factors aid the assembly first of the 30S initiation
complex and then of the 70S initiation complex.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.4. Protein Factors Play Key Roles in Protein Synthesis
Figure 29.28. Structure of Elongation Factor Tu. The structure of a complex between elongation factor Tu (EF-Tu)
and an aminoacyl-tRNA. The amino-terminal domain of EF-Tu is a P-loop NTPase domain similar to those in
other G proteins.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.4. Protein Factors Play Key Roles in Protein Synthesis
Figure 29.29. Molecular Mimicry. The structure of elongation factor G (EF-G) is remarkably similar in shape to that of
the EF-Tu-tRNA complex (see Figure 29.28). The amino-terminal region of EF-G is homologous to EF-Tu, and
the carboxyl-terminal region (shown in red) comprises a set of protein domains that adopted the shape of a tRNA
molecule over the course of evolution.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.4. Protein Factors Play Key Roles in Protein Synthesis
Figure 29.30. Translocation Mechanism. In the GTP form, EF-G binds to the EF-Tu-binding site on the 50S subunit.
This stimulates GTP hydrolysis, inducing a conformational change in EF-G, and driving the stem of EF-G into the A site
on the 30S subunit. To accommodate this domain, the tRNAs and mRNA move through the ribosome by a distance
corresponding to one codon.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.4. Protein Factors Play Key Roles in Protein Synthesis