October 16, 2012

The ribosome influences the accuracy of translation

  • The structure of the 16S rRNA at the P and A sites of the ribosome influences the accuracy of translation.
The lack of detectable variation when the sequence of a protein is analyzed demonstrates that protein synthesis must be extremely accurate. Very few mistakes are apparent in the form of substitutions of one amino acid for another. There are two general stages in protein synthesis at which errors might be made (see Figure 6.8 in 6.3 Special mechanisms control the accuracy of protein synthesis):
  • Charging a tRNA only with its correct amino acid clearly is critical. This is a function of the aminoacyl-tRNA synthetase. Probably the error rate varies with the particular enzyme, but generally mistakes occur in <1/105 aminoacylations.
  • The specificity of codon-anticodon recognition is crucial, but puzzling. Although binding constants vary with the individual codon-anticodon reaction, the specificity is always much too low to provide an error rate of <105. When free in solution, tRNAs bind to their trinucleotide codon sequences only rather weakly. Related, but erroneous, triplets (with two correct bases out of three) are recognized 101102 times as efficiently as the correct triplets.
Codon-anticodon base pairing therefore seems to be a weak point in the accuracy of translation. The ribosome has an important role in controlling the specificity of this interaction, functioning directly or indirectly as a "proofreader," to distinguish correct and incorrect codon-anticodon pairs, and thus amplifying the rather modest intrinsic difference by ~1000×. And in addition to the role of the ribosome itself, the factors that place initiator- and aminoacyl-tRNAs in the ribosome also may influence the pairing reaction.
So there must be some mechanism for stabilizing the correct aminoacyl-tRNA, allowing its amino acid to be accepted as a substrate for receipt of the polypeptide chain; contacts with an incorrect aminoacyl-tRNA must be rapidly broken, so that the complex leaves without reacting. Suppose that there is no specificity in the initial collision between the aminoacyl-tRNA·EF-Tu·GTP complex and the ribosome. If any complex, irrespective of its tRNA, can enter the A site, the number of incorrect entries must far exceed the number of correct entries.
There are two basic models for how the ribosome might discriminate between correctly and incorrectly paired aminoacyl-tRNAs. The actual situation incorporates elements of both models.
  • The direct recognition model supposes that the structure of the ribosome is designed to recognize aminoacyl-tRNAs that are correctly paired. This would mean that the correct pairing results in some small change in the conformation of the aminoacyl-tRNA that the ribosome can recognize. Discrimination occurs before any further reaction occurs.
  • The kinetic proofreading model proposes that there are two (or more) stages in the process, so that the aminoacyl-tRNA has multiple opportunities to disengage. An incorrectly paired aminoacyl-tRNA may pass through some stages of the reaction before it is rejected. Overall selectivity can in principle be the product of the selectivities at each stage.
Figure 7.27 illustrates diagrammatically what happens to correctly and incorrectly paired aminoacyl-tRNAs. A correctly paired aminoacyl-tRNA is able to make stabilizing contacts with rRNA. An incorrectly paired aminoacyl-tRNA does not make these contacts, and therefore is able to diffuse out of the A site.
The path to discovering these interactions started with investigations of the effects of the antibiotic streptomycin in the 1960s. Streptomycin inhibits protein synthesis by binding to 16S rRNA and inhibiting the ability of EF-G to catalyze translocation. It also increases the level of misreading of the pyrimidines U and C (usually one is mistaken for the other, occasionally for A). The site at which streptomycin acts is influenced by the S12 protein; the sequence of this protein is altered in resistant mutants. Ribosomes with an S12 protein derived from resistant bacteria show a reduction in the level of misreading compared with wild-type ribosomes. In effect, S12 controls the level of misreading. When it is mutated to decrease misreading, it suppresses the effect of streptomycin.
S12 stabilizes the structure of 16S rRNA in the region that is bound by streptomycin. The important point to note here is that the P/A site region influences the accuracy of translation: translation can be made more or less accurate by changing the structure of 16S rRNA. The combination of the effects of the S12 protein and streptomycin on the rRNA structure explains the behavior of different mutants in S12, some of which even make the ribosome dependent on the presence of streptomycin for correct translation (Carter et al., 2000).
We now know from the crystal structure of the ribosome that 16S rRNA is in a position to make contacts with aminoacyl-tRNA (for review see Ramakrishnan, 2002). Two bases of 16S rRNA can contact the minor groove of the helix formed by pairing between the anticodon in tRNA with the first two bases of the codon in mRNA (Ogle et al., 2001). This directly stabilizes the structure when the correct codon-anticodon contacts are made at the first two codon positions, but it does not monitor contacts at the third position.
The stabilization of correctly paired aminoacyl-tRNA may have two effects. By holding the aminoacyl-tRNA in the A site, it prevents it from escaping before the next stage of protein synthesis. And the conformational change in the rRNA may help to trigger the next stage of the reaction, which is the hydrolysis of GTP by EF-Tu.
Part of the proofreading effect is determined by timing. An aminoacyl-tRNA in the A site may in effect be trapped if the next stage of protein synthesis occurs while it is there. So a delay between entry into the A site and peptidyl transfer may give more opportunity for a mismatched aminoacyl-tRNA to dissociate. Mismatched aminoacyl-tRNA dissociates more rapidly than correctly matched aminoacyl-tRNA, probably by a factor of ~5×. Its chance of escaping is therefore increased when the peptide transfer step is slowed (for review see Kurland, 1992).
The specificity of decoding has been assumed to reside with the ribosome itself, but some recent results suggest that translation factors influence the process at both the P site and A site. An indication that EF-Tu is involved in maintaining the reading frame is provided by mutants of the factor that suppress frameshifting. This implies that EF-Tu does not merely bring aminoacyl-tRNA to the A site, but also is involved in positioning the incoming aminoacyl-tRNA relative to the peptidyl-tRNA in the P site.
A striking case where factors influence meaning is found at initiation. Mutation of the AUG initiation codon to UUG in the yeast gene HIS4 prevents initiation. Extragenic suppressor mutations can be found that allow protein synthesis to be initiated at the mutant UUG codon. Two of these suppressors prove to be in genes coding for the α and β subunits of eIF2, the factor that binds Met-tRNAi to the P site. The mutation in eIFβ2 resides in a part of the protein that is almost certainly involved in binding nucleic acid. It seems likely that its target is either the initiation sequence of mRNA as such or the base-paired association between the mRNA codon and tRNAiMet anticodon. This suggests that eIF2 participates in the discrimination of initiation codons as well as bringing the initiator tRNA to the P site.
The cost of protein synthesis in terms of high-energy bonds may be increased by proofreading processes. An important question in calculating the cost of protein synthesis is the stage at which the decision is taken on whether to accept a tRNA. If a decision occurs immediately to release an aminoacyl-tRNA·EF-Tu·GTP complex, there is little extra cost for rejecting the large number of incorrect tRNAs that are likely (statistically) to enter the A site before the correct tRNA is recognized. But if GTP is hydrolyzed before the mismatched aminoacyl-tRNA dissociates, the cost will be greater. A mismatched aminoacyl-tRNA can be rejected either before or after the cleavage of GTP, although we do not know yet where on average it is rejected. There is some evidence that the use of GTP in vivo is greater than the three high-energy bonds that are used in adding every (correct) amino acid to the chain