Structural alterations far from the anticodon of the tRNAProGGG of Salmonella typhimurium induce +1 frameshifting at the peptidyl-site.
A total of 12 Salmonella typhimurium mutants were selected with mutations in the minor tRNAProGGG which suppress a +1 frameshift mutation in the hisD gene. This tRNA normally has 1-methylguanosine (m1G37) next to and 3' of the anticodon (position 37). Since the presence of m1G37 prevents frameshifting, some of the +1 frameshift suppressor derivatives of tRNAProGGG had alterations in the primary sequence abolishing the formation of m1G37. However, several of the mutant tRNAProGGG species had a normal level of m1G37 and a normal-sized anticodon loop, showing that neither m1G37 deficiency, nor an oversized anticodon loop, is a prerequisite for +1 frameshifting. Moreover, base substitutions far from the anticodon, e.g. in the acceptor stem, DHU-loop and stem, and at the top of the anticodon stem, promoted +1 frameshifting. When the frameshifting site (CCC-Uaa; CCC is in the zero frame and a +1 frameshift moves the ribosome to the CC-U codon) is overlapped by a nonsense codon (UAA), the efficiency of frameshifting decreased when release factor 1 was over-expressed and increased at an elevated temperature in a mutant with a temperature-sensitive release factor 1. The frameshifting site (CCC-Uac) was also overlapped with the sense codon UAC, which is decoded by a tRNA species having a 2-methylthio-cis ribozeatin (ms2io6A) at position 37. Mutations in the miaA gene affect the formation of this modified nucleoside and result in an A instead of ms2io6A37 in the tRNA. Such an undermodified tRNA is very inefficient in translation and the efficiency of frameshifting increased in a miaA1 mutant. These results suggest that the frameshifting event occurs at the P-site, since the efficiency of frameshifting was sensitive to the decoding activity of the overlapping codon. We conclude that tRNA with mutations far from the anticodon, with a normal-sized anticodon loop and having m1G37 induce +1 frameshifting at the P-site.
Qian
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Department of Microbiology, Umeâ University, Umeâ, S-90 187, Sweden.
Björk
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GR
D000429:Alcohol Oxidoreductases; D000926:Anticodon; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D016368:Frameshift Mutation; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005815:Genetic Code; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D010441:Peptide Chain Elongation, Translational; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics*; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics*; D012361:RNA, Transfer, Pro / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012486:Salmonella typhimurium / Q000235:genetics*; D013696:Temperature
D000926:Anticodon; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D012361:RNA, Transfer, Pro; D011993:Recombinant Fusion Proteins; D006151:Guanosine; C020770:1-methylguanosine; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase