|Content of Protein Synthesis
A. Types of Potential Errors
Given the complexity of protein synthesis, there are many
steps at which mistakes can occur, and it is remarkable
that the error rate is as low as it is. The two principal
categories of translational mistakes are missense errors,
in which one amino acid is substituted for another or a
stop codon is not recognized, and errors of processivity,
in which frameshifting or premature termination occur.
Missense errors can take place because of misacylation
of a tRNA by an AARS, resulting in insertion of an incorrect
amino acid despite a correct codon–anticodon match.
This potential problem is counteracted by active editing
by AARSs (see earlier), which is critical for maintaining
translational accuracy. Errors can also occur at the level of
the codon–anticodon interaction, such that a noncognate
AA–tRNA is selected at the ribosome, again resulting in
amino acid misincorporation. The relative abundance of
cognate vs. noncognate AA–tRNA affects the accuracy
of amino acid incorporation. In particular when the next
amino acid specified by the mRNA is unavailable, the
frequency of missense errors increases. A special case
of incorrect AA–tRNA selection is that of stop codon
readthrough, in which an amino acid is inserted where
the protein should actually be terminated. This type of error
results in a carboxy-terminal extension (added to the
nascent protein) that continues until the ribosome reaches
the next stop signal.
Frameshift mutations occur because of a nucleotide insertion
or deletion in a protein’s gene. Errors in reading
frame also occur when the ribosome “slips” along the
mRNA in such a way that the sequence is not read in
triplets corresponding to the codons of the message, but
the ribosomemoves two or four nucleotides instead. These
“slips” are −1 or +1 frameshifts, respectively. Fortunately
the ribosome usually encounters a stop codon shortly after
the frameshift; this minimizes the effect of the mutation.
(Although frameshifting can be the unintended result of
translational inaccuracy, “programmed” frameshifts also
take place in special situations. These produce alternate
polypeptides from a single mRNA.) Premature termination
of a protein can occur when a nucleotide substitution
produces a stop codon in the middle of the gene sequence.
The peptidyl–tRNAmay also dissociate prematurely from
the ribosome before the stop codon is reached.
B. Suppressor tRNAs
For several of these error scenarios, faithful translation of
the genetic message is accomplished through the action of
suppressor tRNAs. These are the result of advantageous
mutations in tRNA genes, typically in the anticodon, that
allow them to recognize a misplaced stop codon or unintended
frameshift. Suppressor tRNAs may be aminoacylated
according to the amino acid specificity of their
parent sequence, so therefore they insert this amino acid
into the polypeptide at the location of the mRNA aberration.
Alternatively, they may be misacylated because of the
change in the anticodon sequence. As long as the inserted
amino acid is not detrimental to the synthesized protein, a
suppressor tRNA rescues the gene mutation. For example
the E. coli su6 suppressor tRNA inserts leucine into the
growing polypeptide chain at the position of a premature
UAA stop codon.
Missense suppressors simply substitute one amino acid
for another to correct a mutation in the mRNA. Transfer
RNAs that suppress +1 frameshifts typically contain
an extra base in the anticodon and read a four-nucleotide
codon, thereby restoring the correct translational frame.
Suppressor tRNAs are of particular interest as more examples
of the genetic basis for specific diseases are found.
In many cases, missense or nonsense mutations in genes
for essential proteins correlate with diseases such as cancer,
cystic fibrosis, and amyotropic lateral sclerosis (ALS).
In fact, mutations in the tumor suppressor gene p53 are the
single greatest cause of human cancers. One particular p53 hotspot is at codon 213, where nonsense mutations have
been identified in human colorectal, gastric, ovarian, and
breast cancers. Suppressor tRNAs designed to selectively
rescue these mutations by reading through the premature
termination codon could be effective therapeutic agents
in a gene therapy approach. Several challenges remain,
however—the suppressor tRNA must be introduced into
the affected cells, must be transcribed at high enough levels
to be effective, must also be aminoacylated at high
levels, and must not excessively read through stop codons
that specify the normal termination of translation for
other genes. These are active areas of research in several
C. Selenocysteine Insertion at UGA Codons
The trace element selenium is present in a number
of proteins in the form of cotranslationally inserted
selenocysteine (Sec). This insertion occurs when a unique
selenocysteinyl–tRNASec recognizes an internal UGA
codon. Selenocysteine has therefore been called the “21st
amino acid” and its insertion at a nucleotide triplet that is
normally a termination signal represents an expansion of
the genetic code.
Insertion of selenocysteine requires several adaptations
of the translational machinery. First, tRNASec is aminoacylated
with serine by seryl–tRNA synthetase. The serine
attached to this misacylated species is then converted
to selenocysteine by the enzyme selenocysteine synthase,
which uses selenophosphate as a donor. Elongation
factor Tu does not bind Sec–tRNASec as it does other
elongator AA–tRNAs; instead, a unique protein SELB
transports Sec-tRNASec to the ribosomal A-site. SELB is
specific for Sec–tRNASec, rejecting other AA–tRNAs
including Ser–tRNASec. The final novel feature of selenocysteine
insertion is the mechanism ofmRNArecognition.
The UGA triplet can be used in the same organism as
either a selenocysteine or a stop codon. The sequence
context determines its recognition as a selenocysteine
codon. The ternary complex SELB:GTP:Sec–tRNASec recognizes a stem-loop structure immediately 3´ (downstream)
from a UGA codon that is read as selenocysteine.
This structural feature of the mRNA is specifically
bound by the carboxyl-terminal portion of SELB, while
other regions of the protein are highly homologous
to EF-Tu as expected. Insertion of selenocysteine into
polypeptides therefore requires formation of a quaternary
SELB:GTP:Sec–tRNASec:mRNA complex. This is in
contrast to all other elongation steps in protein synthesis,
which proceed through ternary complexes.
D. Degradation of Incomplete Polypeptides
One consequence of the necessary accuracy in protein synthesis
is the release of peptidyl–tRNA molecules representing
incomplete translation products. These products
can be the result of ribosome stalling, a premature stop
codon that is not suppressed, or detection by the ribosome
of noncognate tRNA present in the decoding center. It has
been estimated that this “drop-off” might result in a truncated
polypeptide chain approximately 10% as often as the
full-length protein. Not only is this a waste of amino acids
resulting in useless products but also tRNA molecules are
sequestered and unavailable for translation of other genes.
The incomplete peptidyl–tRNAs are substrates in bacteria
and yeast for the enzyme peptidyl–tRNA hydrolase,
which cleaves the ester bond between the tRNA and its
attached polypeptide. Peptidyl–tRNA hydrolase therefore
removes the useless (and potentially harmful) protein fragments
and recycles the tRNAs. Interestingly, although the
initiator tRNA(fMet–tRNAfMet) mimics a peptidyl–tRNA
by virtue of its N-formyl group, peptidyl–tRNAhydrolase
does not recognize the initiator as a substrate. The hydrolase
bypasses fMet–tRNAfMet because of the presence of
structural features unique to fMet–tRNAfMet.
E. A Dual-Function RNA
A particular challenge arises when an mRNA lacks inframe
stop codons due to deletion or degradation. Bacteria
use a unique tRNA–mRNA hybrid (tmRNA, also called
10Sa RNA) to remove the resulting partially synthesized
proteins and free the ribosomal subunits. The 5´- and
3´-ends of this molecule resemble alanyl–tRNA, while
the central portion encodes a peptide “tag.” Alanyl–tRNA
synthetase aminoacylates the tmRNA, which is then
transported to the stalled elongation complex by EF-Tu.
After attaching alanine to the end of the growing nascent
polypeptide, the ribosome switches from the truncated
mRNA to the tmRNA in a mechanism called transtranslation.
The ribosome adds 10 additional amino acids
to the end of the protein according to the tmRNA sequence.
The resulting tagged protein is released and degraded, as
the tag is a recognition signal for several proteases.
F. mRNA Surveillance
Eukaryotes use a surveillance mechanism to identify
mRNAs with mutations (typically premature termination
codons) or processing errors (such as incorrect splicing).
Once detected, the aberrant messages are degraded
to prevent the synthesis of truncated proteins. Recent
evidence suggests that these mRNAs must be at least
partially translated to determine whether a stop codon is in
its proper context. In mammalian systems, the translating
ribosome is proposed to measure the distance between
the final splicing junction and the termination signal—if
they are within 50 nucleotides of one another, termination
is allowed to proceed. If they are further apart, either
because of a misplaced stop codon within the mRNA
reading frame or a splicing error, the mRNA is targeted
for rapid degradation. How the ribosome recognizes this
distance is not yet known. This mRNA surveillance is also
called nonsense-mediated decay because the majority of
mutational errors result in premature termination codons.
G. Accuracy Mechanisms
Despite the types of translational errors described above,
mRNA-directed protein synthesis is remarkably accurate.
How is it that the ribosome and translational factors are
able to achieve such faithful transmission of genetic information?
One way to describe the specificity of cognate
over noncognate AA–tRNAs is termed the “kinetic
proofreading” mechanism. One can imagine that selection
of an EF-Tu:GTP:AA–tRNA ternary complex by the
ribosome during elongation can be considered a “scanning”
step. Depending on the codon–anticodon interaction,
the AA–tRNA will either bind irreversibly in the ribosomal
A-site (in the case of the cognate AA–tRNA), or
dissociate from the ribosome before or after GTP hydrolysis
(noncognate AA–tRNA). In this model, the rate of
EF-Tu-triggered GTP hydrolysis is the same for cognate
and noncognate substrates. However, because the cognate
AA–tRNA spends more time in complex with the ribosome,
GTP hydrolysis is likely to occur prior to dissociation
of the tRNA, promoting tight binding of this tRNA
in the A-site prior to peptide bond formation.
In addition to passive kinetic proofreading, selection
of the correct codon–anticodon interaction is proposed to
trigger a conformational change in the ribosome that is
transmitted to EF-Tu. Hydrolysis of GTP is then accelerated
for the cognate compared to the noncognate substrate.
Recent structural evidence shows that structural rearrangements
do occur upon binding of the cognate AA–tRNA,
suggesting that selection of the correct substrate depends
on an induced fit mechanism. How the codon–anticodon
interaction is detected by the ribosome is not currently
H. Ribosomal Contributions to Accuracy
Naturally occurring mutations have been identified in ribosomal
proteins and rRNA that increase or decrease translational
accuracy. These have provided clues as to which
regions of the ribosome are involved in proofreading, although
detailed mechanisms are not known. The small
subunit, which contains the decoding site, has several proteins
that are likely involved in controlling missense and
processivity errors. For example, mutations have been
identified in proteins S4 and S5 that reduce the level of
translational fidelity (these are called ribosomal ambiguity
mutations). In contrast, S12 mutants increase the accuracy
of translation by conferring resistance to streptomycin, an
The small subunit 16S rRNA also contains regions that
appear to be involved in translational accuracy. In particular,
substitutions at nucleotides in the so-called 530
loop lead to changes in the error rate of protein synthesis.
This rRNA loop is known to be spatially near the proteins
S4, S5, and S12, and effects of nucleotide changes in the
530 loop parallel those observed for the proteins. Thus,
the rRNA and proteins in this part of the small subunit
together act as a proofreading domain. Substitutions at
some of these nucleotides increase the rate of missense
or frameshift errors, while others are detrimental because
they prevent binding of the EF-Tu:GTP:AA–tRNAternary
complex. Still other mutations actually increase the accuracy
of translation, in that they make the ribosomes resistant
to error-inducing antibiotics.
The toxins α-sarcin and ricin inactivate ribosomes by
altering a highly conserved sequence in 23S-like rRNAs
(the sarcin–ricin loop). Elongation factors Tu and G bind
near this loop, suggesting that it is involved in AA–tRNA
selection and translocation. Protein L6 and the sarcin–
ricin loop are involved in translational accuracy control,
as suggested by the mutations in these components that
increase fidelity, apparently by slowing down translation
to allow more thorough proofreading.