MOLECULAR BASIS OF TRANSLATIONAL RECODING IN YEAST

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This proposal concerns the mechanism of +1 translational frameshifting, both events which occur at special mRNA sites which promote a shift in reading frame (programmed frameshifting) and phenotypic suppression of frameshift mutations caused by mutant tRNAs (frameshift suppression). It has long been thought that these two events were unrelated mechanistically. The classical model for frameshift suppression proposes that a mutant tRNA with an expanded anticodon loop reads an expanded four nucleotide anticodon, causing a shift in the downstream or +1 direction. Programmed frameshifts by contrast now appear to occur because of near-cognate decoding of the last in-frame codon, which leads to a translational error at the next codon shifting reading. The classical model of suppression now appears invalid, and suppression may occur by a mechanism similar to programmed frameshifts. This proposal seeks to further explore the mechanism of programmed frameshifting and to test whether the near-cognate decoding model for frameshift suppression is correct. Yeast molecular genetic analysis will be used to test further the validity of the near-cognate decoding model for programmed frameshifting. PCR-based gene disruption will eliminate each of the low-copy tRNA genes in the yeast genome; in such strains the cognate codons must be read by a near-cognate isoacceptor, which may induce high efficiency frameshifting. In some cases frameshifting occurs because the normal cognate tRNA decodes doo inefficiently; site-specific mutagenesis will identify the sequences in the tRNAs contributing to their inefficiency. An in vitro translation system which promotes +1 programmed frameshifting will be used to identify which near-cognate tRNAs are actually responsible for promoting the shift in frame. Finally, the basis of frameshift stimulation by a cis-acting mRNA sequence, and by mutants of elongation factor-1alpha will be assessed using site-specific mutagenesis and classical genetic analysis. Key features of a new model for phenotypic suppression of frameshift mutations will be tested in the proposal. First, the necessity for a four nucleotide interaction between the tRNA and mRNA will be tested by site-specific mutagenesis of a gene encodig a frameshift suppressor tRNA. Second, the proposal will test the prediction that expansion of the anticodon loop stabilizes near-cognate decoding, and destabilizes cognate decoding. Third, it will test whether expansion only stimulates frameshifting by tRNAs which can slip on the mRNA. Finally, it will test if the same mechanism governs suppression of -1 frameshift mutations.