The Efficiency of Translation Termination is Determined by a Synergistic Interplay Between Upstream and Downstream Sequences inSaccharomyces cerevisiae

Comprehensive Analysis of Stop Codon Usage in Bacteria and Its Correlation with Release Factor Abundance

“…In the few coding sequences available at that time, TAA was observed to be the most abundant stop codon. The authors concluded that this strong bias toward TAA might be due to the fact that it can be recognized by both release factors, which was suggested in other related studies as well (9,10). In a later work, Sun et al (11) analyzed more than 70,000 genes from eukaryotes including fungi, plant, and human.…”

“…In a more detailed investigation, stop codon determinants in six prokaryotic and five eukaryotic genomes were studied with the same conclusions (12). In other earlier works, the context of surrounding nucleotides, especially those immediately after the stop codon, in translation termination has been discussed (10,13,14). Also, tools such as TRANSTERM were prepared to investigate the up-and downstream regions of stop codons in a given species (9).…”

Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment

“…12,13,15,26,31,32 A C residue following either a UAA or a UGA stop codon has been reported to be usually correlated with high levels of readthrough, either in the presence or absence of an aminoglycoside antibiotic. UAG gave more erratic results, with U or C being associated with high or low readthrough, possibly reflecting the difference in the surrounding context between the experimental systems used.…”

“…11 Secondly, several studies demonstrated the critical influence of both upstream and downstream sequences, which determine the overall efficiency of translational termination in yeast. 12,13 Less is currently known on the influence of the sequence context on basal and antibiotic-induced readthrough levels in mammalian cells. Results from different laboratories have demonstrated that the antibiotic effect is dependent on the nucleotides surrounding the stop mutation; they also suggest that the nucleotide immediately following the stop codon (numbered þ 4) has a major role in readthrough.…”

Terminating eukaryote translation: Domain 1 of release factor eRF1 functions in stop codon recognition

“…Two explanations are offered; the first is that such eRF1 mutants might be strong suppressors that may not support yeast viability and so were not isolated+ A second explanation is that stop codon FIGURE 6. Western blot analysis of ribosome-bound eRF1 and eRF3 in the eRF1 mutant strains+ Ribosomal fractions were prepared from the sup45-disruptant strain IS37/7b supported by either the wild-type SUP45 ϩ allele, or each of the mutant alleles+ Proteins (25 mg) were separated using SDS-PAGE, and either stained with Coomassie Blue to verify equivalent loadings (A), or blotted onto nitrocellulose and probed with anti-eRF3 antibody (B), or anti-eRF1 antibody (C)+ Antibody binding was visualized using secondary antibody and chemiluminescent detection (see Materials and methods)+ The migration positions of molecular mass standards are marked, as are western blot bands corresponding to eRF1 (49 kDa) and eRF3 (79 kDa)+ The second, heavier (70 kDa) band on the eRF1 western blot is routinely seen using this affinity purified antibody preparation (Stansfield et al+, 1992)+ Western blot samples are split between blot 1 and blot 2, each with its own SUP45 ϩ control+ recognition by eRF1 might be "holistic"; in other words, a subtle interplay between eRF1 amino acids comprising a stop codon binding site achieves cooperative codon recognition, and a mutation affecting recognition of one stop codon inevitably has consequences for recognition of the other two+ All the mutants exhibit increased UAA suppression to varying degrees+ However, we believe our estimates of the true abilities of the mutant eRF1s to recognize UAA were distorted by the presence of the SUQ5 suppressor tRNA present in the IS37/7b strain background; SUQ5, unlike the cell's population of natural suppressor tRNAs, is cognate for UAA, and miscognate for UAG, and so will artificially increase suppression of UAA, and to a lesser extent UAG, above that which would be detected in a wild-type tRNA background+ In addition to stop-codon-suppression biased eRF1 alleles, the screens also identified the omnipotent suppressors sup45-702, sup45-718, and sup45-731, although these mutants phenotypically suppressed only the ade1-14 mutation+ Presumably these mutants suppressed lys2-864 UAG and his7-1 UAA stop codons weakly+ Curiously, mutants 702, 718, and 731 also show very low levels of UGA suppression (measured with the lacZ reporter system) despite being isolated as ade1-14 suppressors+ This is not because the ade1-14 premature stop codon is in an easily-suppressed nucleotide context, although context does have important effects on stop recognition by eRF1 in yeast (Bonetti et al+, 1995;Mottaguitabar et al+, 1998)+ Sequence analysis of the premature ade1-14 UGA stop codon identified the mutated codon as TGG (Trp 244) r TGA in the context TTC TGA AAC (data not shown), which defines a reasonably good eRF1 substrate (Bonetti et al+, 1995;Mottaguitabar et al+, 1998)+…”