To insure survival, cells must accurately and efficiently translate information encoded in messenger RNA (mRNA) sequences into proteins. Maximizing accuracy and efficiency puts conflicting demands on the translational machinery. The resolution of these conflicting demands has involved the evolution of multiple accuracy mechanisms. Despite these mechanisms, errors do occur including missense errors, frameshifting errors and errors involving release of the peptidyl-tRNA from the ribosome (translational drop-off).
Estimates of the frequency of these errors vary widely. This is especially true for missense errors, which are estimated to occur at rates of from 10-2 to 10-4 per codon in vivo. Estimate variation probably results from the fact that different studies measured qualitatively different errors (errors at the first, second or third codon position), studied errors by different tRNAs and at different codons, and used different methods.
No published study had attempted to estimate all types of error rates by a single tRNA. We have devised a method using mutations affecting essential active site residues of reporter genes. We have shown that the excess residual activity of some mutations results from misreading of the mutant codon as the wild type amino acid. For example, we have shown that mutations replacing Lys-529 of Photinus (firefly) luciferase with an Arg encoded by either AGA or AGG results in a high residual activity. Since replacing the Lys-529 codon with the Arg codons CGU, CGC, CGA or CGG resulted in much less activity, we conclude that the high residual activity of the AGA or AGG mutants results from misreading by tRNALys involving a middle position U•G mismatch.
By studying all possible errors by tRNALys at codon 529 we have shown that the frequency of error varies by over an order of magnitude. The strongest determinant of error rate is the availability of the cognate tRNA that competes for tRNALys at the codon. The fact that AGA and AGG-decoding tRNAArg is in very low abundance explains the high frequency of error at the codon.
We have used the system to study the effect of error-inducing antibiotics (streptomycin and paromomycin) on error frequency finding that their effect is very codon-specific. We have also tested the effect of mutants of ribosomal proteins rpS12 and rpS4, which increase and decrease accuracy, respectively. Again, their effect is very codon-specific. In each case only a small subset of codons is strongly affected, mostly those with low abundance cognate tRNAs.
We are extending this work to look at error rates of various tRNAs and at various of codons. Preliminary data show that error rate vary even more widely when these are taken into consideration.