Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

Winey, Mark; Mendenhall, Michael J.; Cummins, Claudia M.; Culbertson, Michael R.; Knapp, Gayle

doi:10.1016/0022-2836(86)90463-8

Cited by 31 publications

(17 citation statements)

References 29 publications

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“…Equimolar yields of the 5' half-tRNA, 3' halftRNA, and intron were obtained. The identities of these products were determined previously (43). The reaction products reached near maximum levels of accumulation by 30 min, at which time approximately 20 to 30% of the precursor had been utilized.…”

Section: Resultsmentioning

confidence: 91%

“…One allele has been analyzed in greater detail to determine the exact sites of endonuclease cleavage. It was shown by RNA sequence analysis that the mature, in vivo product of the SUF8-1 gene (group C, C31-U39) is produced as the result of cleavages at the same 5' and 3' sites as in the wild-type tRNA (43). Furthermore, a comparison of SUF8-1 alleles either containing or lacking an intron suggested that the tRNA product derived by splicing acts as the sole agent of suppression.…”

Section: Discussionmentioning

confidence: 99%

“…Cleavage assays were performed as described by Winey et al (43), using 0.1 U of enzyme per ml and approximately 1.5 x 108 g of pre-tRNA (30 nM). The pre-tRNAs remained intact, and no cleavage products were observed in reaction mixtures containing 1% Triton X-100 but lacking the fraction IV extract.…”

Section: Methodsmentioning

confidence: 99%

“…The his4-713 mutation in strain W153-lOb causes a shift in the his4 message to the -1 reading frame as a result of a + 1C nucleotide insertion in a CCU proline codon (7 (43). The assignments and amounts of products formed were also determined by 3' end labeling (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…SUF8 pre-tRNA was chosen for this analysis in part because its secondary structure has been described and conforms to the structural consensus typical of other S. cerevisiae pre-tRNAs (17). In addition, a previous study showed that the mutation SUF8-1 (G39-*U39) reduces the efficiency of in vitro cleavage by disrupting the secondary structure of the anticodon stem (43).…”

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confidence: 99%

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Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae.

Mathison¹,

Winey²,

Soref³

et al. 1989

Mol. Cell. Biol.

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To evaluate the role of exon domains in tRNA splicing, the anti-codon stem of proline pre-tRNAUGG from Saccharomyces cerevisiae was altered by site-directed mutagenesis of the suJ8 gene. Sixteen alleles were constructed that encode mutant pre-tRNAs containing all possible base combinations in the last base pair of the anticodon stem adjacent to the anticodon loop (positions 31 and 39). The altered pre-tRNAs were screened by using an in vitro endonucleolytic cleavage assay to determine whether perturbations in secondary structure affect the intron excision reaction. The pre-tRNAs were cleaved efficiently whenever secondary structure in the anticodon stem was maintained through standard base pairing or G-U interactions. However, most of the pre-tRNAs with disrupted secondary structure were poor substrates for intron excision. We also determined the extent to which the suJ8 alleles produce functional products in vivo. Each allele was integrated in one to three copies into a yeast chromosome or introduced on a high-copy-number plasmid by transformation. The formation of a functional product was assayed by the ability of each allele to suppress the + 1 frameshift mutation his4-713 through four-base codon reading, as shown previously for the SUF8-1 suppressor allele. We found that alleles containing any standard base pair or G U pair at position 31/39 in the anticodon stem failed to suppress his4-713. We could not assess in vivo splicing with these alleles because the tRNA products, even if they are made, would be expected to read a normal triplet rather than a quadruplet codon. However, all of the alleles that contained a disrupted base pair at position 31/ 39 in the anticodon stem altered the structure of the tRNA in a manner that caused frameshift suppression. Suppression indicated that splicing must have occurred to some extent in vivo even though most of the suppressor alleles produced pre-tRNAs that were cleaved with low efficiency or not at all in vitro. These results have important implications for the interpretation of in vitro cleavage assays in general and for the potential use of suppressors to select mutations that affect tRNA splicing.Splicing of transfer RNA in Saccharomyces cerevisiae requires three separate enzymes: a detergent-extractable endonuclease, a soluble ligase, and a 2'-phosphatase (26). The endonuclease cleaves the pre-tRNA, releasing the intron as a linear molecule (25). The ligase joins the half molecules through an ATP-dependent, three-step mechanism involving cyclic phosphodiesterase, polynucleotide kinase, and RNA ligase activities (12, 28). Finally, the residual 2'-phosphate at the splice site is removed to produce a mature, primary tRNA sequence.The splicing enzymes of S. cerevisiae process all introncontaining intermediates in the synthesis of 10 of the 46 cytoplasmic tRNA isoacceptors (14, 23). The enzymes form an in vitro complex composed minimally of the endonuclease and the ligase (11). The ligase, and by extension probably the complex as well, is localized in vivo in associatio...

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Section: Resultsmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae.

Mathison¹,

Winey²,

Soref³

et al. 1989

Mol. Cell. Biol.

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Mutants of elongation factor Tu promote ribosomal frameshifting and nonsense readthrough.

1987

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This is the first report of ribosomal frameshifting promoted by mutants of the elongation factor Tu (EF‐Tu). EF‐Tu mutants can suppress both −1 and +1 frameshift mutations. The level of nonsense readthrough is also increased at some UGA (this paper) and UAG (Hughes, 1987) sites by these mutants. Suppression occurs when a mutant tuf allele is paired with a wild‐type copy of the other tuf gene but is most efficient when both tuf genes are mutant. Frameshifting mediated by the tuf alleles studied, tufA8 and tufB103, is not general; indeed most frameshift mutations are not suppressed. Several possible mechanisms by which mutant EF‐Tu may cause frameshifting are discussed.

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A novel type of + 1 frameshift suppressor: a base substitution in the anticodon stem of a yeast mitochondrial serine-tRNA causes frameshift suppression.

et al. 1990

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We have identified a spontaneous mitochondrial mutation, mfs‐1 (mitochondrial frameshift suppressor‐1), which suppresses a + 1 frameshift mutation localized in the yeast mitochondrial oxi1 gene. The suppressor strain exhibits a single base change (C to U) at position 42 of the mitochondrial serine‐tRNA (UCN). To our knowledge, this is the first reported case showing that a mutation in the anticodon stem of a tRNA can cause frameshift suppression. The expression and aminoacylation of the mutant tRNASer(UCN) are not significantly affected. However, the base change at position 42 has two effects: first, residue U27 of the mutant tRNA is not modified to pseudouridine as observed in wild‐type tRNASer(UCN). Second, the base change and/or the lack of modification of U27 leads to an alteration in the secondary/tertiary structure of the mutant tRNA. It is possible that there are such structural changes in the anticodon loop that enable the tRNA to read a four base codon, UCCA, thus restoring the wild‐type reading frame.

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Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

Cited by 31 publications

References 29 publications

Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae.

Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae.

Mutants of elongation factor Tu promote ribosomal frameshifting and nonsense readthrough.

A novel type of + 1 frameshift suppressor: a base substitution in the anticodon stem of a yeast mitochondrial serine-tRNA causes frameshift suppression.

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