Translon: a single term for translated regions.
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Letter↗
Biomedical subjects
Philip J Farabaugh
Publications and source records attributed to Philip J Farabaugh.
The frequency of translational misreading errors in E. coli is largely determined by tRNA competition.
Estimates of missense error rates (misreading) during protein synthesis vary from 10(-3) to 10(-4) per codon. The experiments reporting these rates have measured several distinct errors using several methods and reporter systems. Variation in reported rates may reflect real differences in rates among the errors tested or in sensitivity of the reporter systems. To develop a more accurate understanding of the range of error rates, we developed a system to quantify the frequency of every possible misreading error at a defined codon in Escherichia coli. This system uses an essential lysine in the active site of firefly luciferase. Mutations in Lys529 result in up to a 1600-fold reduction in activity, but the phenotype varies with amino acid. We hypothesized that residual activity of some of the mutant genes might result from misreading of the mutant codons by tRNA(Lys) (UUUU), the cognate tRNA for the lysine codons, AAA and AAG. Our data validate this hypothesis and reveal details about relative missense error rates of near-cognate codons. The error rates in E. coli do, in fact, vary widely. One source of variation is the effect of competition by cognate tRNAs for the mutant codons; higher error frequencies result from lower competition from low-abundance tRNAs. We also used the system to study the effect of ribosomal protein mutations known to affect error rates and the effect of error-inducing antibiotics, finding that they affect misreading on only a subset of near-cognate codons and that their effect may be less general than previously thought.
Binding Sites↗
Evolution of +1 programmed frameshifting signals and frameshift-regulating tRNAs in the order Saccharomycetales.
Programmed translational frameshifting is a ubiquitous but rare mechanism of gene expression in which mRNA sequences cause the translational machinery to shift reading frames with extreme efficiency, up to at least 50%. The mRNA sequences responsible are deceptively simple; the sequence CUU-AGG-C causes about 40% frameshifting when inserted into an mRNA in the yeast Saccharomyces cerevisiae. The high efficiency of this site depends on a set of S. cerevisiae tRNA isoacceptors that perturb the mechanism of translation to cause the programmed translational error. The simplicity of the system might suggest that it could evolve frequently and perhaps be lost as easily. We have investigated the history of programmed +1 frameshifting in fungi. We find that frameshifting has persisted in two structural genes in budding yeasts, ABP140 and EST3 for about 150 million years. Further, the tRNAs that stimulate the event are equally old. Species that diverged from the lineage earlier both do not employ frameshifting and have a different complement of tRNAs predicted to be inimical to frameshifting. The stability of the coevolution of protein coding genes and tRNAs suggests that frameshifting has been selected for during the divergence of these species.
Amino Acid Sequence↗
An mRNA sequence derived from a programmed frameshifting signal decreases codon discrimination during translation initiation.
Sequences and structures in the mRNA can alter the accuracy of translation. In some cases, mRNA secondary structures like hairpin loops or pseudoknots can cause frequent errors of translational reading frame (programmed frameshifting) or misreading of termination codons as sense (nonsense readthrough). In other cases, the primary mRNA sequence stimulates the error probably by interacting with an element of the ribosome to interfere with error correction. One such primary mRNA sequence, the Ty3 stimulator, increases programmed +1 frameshifting 7.5 times in the yeast Saccharomyces cerevisiae. Here we show that this stimulator also increases the usage of non-AUG initiation codons in the bacterium Escherichia coli but not in S. cerevisiae. These data suggest that in E. coli, though not in yeast, an element of the ribosome's elongation accuracy mechanism ensures initiation accuracy.
Base Sequence↗
Distinct paths to stop codon reassignment by the variant-code organisms Tetrahymena and Euplotes.
The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed that changes in domain 1 of ciliate eRF1s are responsible for altered stop codon recognition, this has never been demonstrated in vivo. To carry out such an analysis, we made hybrid proteins that contained eRF1 domain 1 from either Tetrahymena thermophila or Euplotes octocarinatus fused to eRF1 domains 2 and 3 from Saccharomyces cerevisiae. We found that the Tetrahymena hybrid eRF1 efficiently terminated at all three stop codons when expressed in yeast cells, indicating that domain 1 is not the sole determinant of stop codon recognition in Tetrahymena species. In contrast, the Euplotes hybrid facilitated efficient translation termination at UAA and UAG codons but not at the UGA codon. Together, these results indicate that while domain 1 facilitates stop codon recognition, other factors can influence this process. Our findings also indicate that these two ciliate species used distinct approaches to diverge from the universal genetic code.
Animals↗
Translational accuracy during exponential, postdiauxic, and stationary growth phases in Saccharomyces cerevisiae.
When the yeast Saccharomyces cerevisiae shifts from rapid growth on glucose to slow growth on ethanol, it undergoes profound changes in cellular metabolism, including the destruction of most of the translational machinery. We have examined the effect of this metabolic change, termed the diauxic shift, on the frequency of translational errors. Recoding sites are mRNA sequences that increase the frequency of translational errors, providing a convenient reporter of translational accuracy. We found that the diauxic shift causes no overall change in translational accuracy but does cause a strong reduction in the frequency of one type of programmed error: Ty +1 frameshifting. Genetic data suggest that this effect may be due to changes in the relative amounts of tRNA participating in translation elongation. We discuss possible implications for expression strategies that use recoding.
Codon↗
Transfer RNA modifications that alter +1 frameshifting in general fail to affect -1 frameshifting.
Using mutants (tgt, mnmA(asuE, trmU), mnmE(trmE), miaA, miaB, miaE, truA(hisT), truB) of either Escherichia coli or Salmonella enterica serovar Typhimurium and the trm5 mutant of Saccharomyces cerevisiae, we have analyzed the influence by the modified nucleosides Q34, mnm(5)s(2)U34, ms(2)io(6)A37, Psi39, Psi55, m(1)G37, and yW37 on -1 frameshifts errors at various heptameric sequences, at which at least one codon is decoded by tRNAs having these modified nucleosides. The frequency of -1 frameshifting was the same in congenic strains only differing in the allelic state of the various tRNA modification genes. In fact, in one case (deficiency of mnm(5)s(2)U34), we observed a reduced ability of the undermodified tRNA to make a -1 frameshift error. These results are in sharp contrast to earlier observations that tRNA modification prevents +1 frameshifting suggesting that the mechanisms by which -1 and +1 frameshift errors occur are different. Possible mechanisms explaining these results are discussed.
Anticodon↗
Shifty ciliates: frequent programmed translational frameshifting in euplotids.
Recent work suggests that there is a high frequency of programmed +1 translational frameshifting in ciliates of the Euplotes genus. Frequent frameshifting may have been potentiated by stop codon reassignment, which is also a feature of this group.
Animals↗
Ribosome structure: revisiting the connection between translational accuracy and unconventional decoding.
The ribosome is a molecular machine that converts genetic information in the form of RNA, into protein. Recent structural studies reveal a complex set of interactions between the ribosome and its ligands, mRNA and tRNA, that indicate ways in which the ribosome could avoid costly translational errors. Ribosomes must decode each successive codon accurately, and structural data provide a clear indication of how ribosomes limit recruitment of the wrong tRNA (sense errors). In a triplet-based genetic code there are three potential forward reading frames, only one of which encodes the correct protein. Errors in which the ribosome reads a codon out of the normal reading frame (frameshift errors) occur less frequently than sense errors, although it is not clear from structural data how these errors are avoided. Some mRNA sequences, termed programmed-frameshift sites, cause the ribosome to change reading frame. Based on recent work on these sites, this article proposes that the ribosome uses the structure of the codon-anticodon complex formed by the peptidyl-tRNA, especially its wobble interaction, to constrain the incoming aminoacyl-tRNA to the correct reading frame.
Protein Biosynthesis↗