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Effect of Intron Mutations on Processing and Function of Saccharomyces cerevisiae SUP53 tRNA In Vitro and In Vivo

Strobel, Marjorie C. and Abelson, John (1986) Effect of Intron Mutations on Processing and Function of Saccharomyces cerevisiae SUP53 tRNA In Vitro and In Vivo. Molecular and Cellular Biology, 6 (7). pp. 2663-2673. ISSN 0270-7306. PMCID PMC367823. doi:10.1128/MCB.6.7.2663.

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The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.

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Additional Information:© 1986 American Society for Microbiology. Received 4 December 1985. Accepted 2 April 1986. We thank Andy Newman, Peter Johnson, Chris Greer, and Eric Phizicky for their advice and encouragement; Zenaida Resplandor for technical assistance; and Linda Brubaker, Connie Katz, and Donna Walker for preparation of the manuscript. This work was supported by American Cancer Society grant ACS NP302D and Public Health Service grant GM-32637 from the National Institutes of Health to J .A. M.C.S. was supported by a Public Health Service Genetics Training Grant (Biology Department, University of California, San Diego).
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American Cancer Society (ACS)NP302D
NIH Public Health Service GrantGM-32637
Public Health Service Genetics Training GrantUNSPECIFIED
Issue or Number:7
PubMed Central ID:PMC367823
Record Number:CaltechAUTHORS:20120627-135342826
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Official Citation:Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo. M C Strobel and J Abelson Mol. Cell. Biol. July 1986 6:2663-2673; doi:10.1128/MCB.6.7.2663
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:32152
Deposited By: Ruth Sustaita
Deposited On:27 Jun 2012 21:10
Last Modified:09 Nov 2021 21:25

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