Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo

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.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  E. Phizicky,et al.  Saccharomyces cerevisiae tRNA ligase. Purification of the protein and isolation of the structural gene. , 1986, The Journal of biological chemistry.

[3]  Phillip A. Sharp,et al.  On the origin of RNA splicing and introns , 1985, Cell.

[4]  D. Söll,et al.  Mutations preventing expression of sup3 tRNASer nonsense suppressors of Schizosaccharomyces pombe , 1985, Molecular and cellular biology.

[5]  H. Domdey,et al.  A test for intron function in the yeast actin gene , 1985, Nature.

[6]  G. Knapp,et al.  Transfer RNA splicing in Saccharomyces cerevisiae. Secondary and tertiary structures of the substrates. , 1985, The Journal of biological chemistry.

[7]  D. Engelke,et al.  Nucleolytic processing of a tRNAArg-tRNAAsp dimeric precursor by a homologous component from Saccharomyces cerevisiae. , 1985, The Journal of biological chemistry.

[8]  R. Ogden,et al.  Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates. , 1984, Nucleic acids research.

[9]  C. Reed,et al.  Yeast amber suppressors corresponding to tRNA3Leu genes. , 1984, Journal of molecular biology.

[10]  D. Söll,et al.  Mutations affecting excision of the intron from a eukaryotic dimeric tRNA precursor. , 1984, The EMBO journal.

[11]  H. Swerdlow,et al.  Structure of intron-containing tRNA precursors. Analysis of solution conformation using chemical and enzymatic probes. , 1984, The Journal of biological chemistry.

[12]  M. Olson,et al.  Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene , 1984, Molecular and cellular biology.

[13]  C. Greer,et al.  RNA splicing: rearrangement of RNA sequences in the expression of split genes , 1984 .

[14]  R. Waterston,et al.  The yeast cloning vector YEp13 contains a tRNALeu3 gene that can mutate to an amber suppressor. , 1984, Gene.

[15]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations. , 1984, Journal of bacteriology.

[16]  J. Mao,et al.  Six Schizosaccharomyces pombe tRNA genes including a gene for a tRNALys with an intervening sequence which cannot base-pair with the anticodon. , 1983, Nucleic acids research.

[17]  R. Ogden,et al.  tRNA gene transcription in yeast: Effects of specified base substitutions in the intragenic promoter , 1983, Cell.

[18]  G. Tocchini-Valentini,et al.  Role of RNA structure in splicing: Excision of the intervening sequence in yeast tRNA3leu is dependent on the formation of a D stem , 1983, Cell.

[19]  T. Cech.  RNA splicing: Three themes with variations , 1983, Cell.

[20]  G. J. Raymond,et al.  The role of non-coding DNA sequences in transcription and processing of a yeast tRNA. , 1983, Nucleic acids research.

[21]  C. Greer,et al.  RNA ligase in bacteria: Formation of a 2′,5′ linkage by an E. coli extract , 1983, Cell.

[22]  J. Abelson,et al.  The yeast tRNATyr gene intron is essential for correct modification of its tRNA product , 1983, Nature.

[23]  J. Miller,et al.  Effects of surrounding sequence on the suppression of nonsense codons. , 1983, Journal of molecular biology.

[24]  J. Abelson,et al.  Mechanism of action of a yeast RNA ligase in tRNA splicing , 1983, Cell.

[25]  J. Abelson,et al.  Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease , 1983, Cell.

[26]  G. Tocchini-Valentini,et al.  Mutation in the A block of the yeast tRNA3 Leu gene that allows transcription but abolishes splicing and 5′-end maturation , 1983, Cell.

[27]  R. Koski,et al.  An in vitro RNA polymerase III system from S. cerevisiae: effects of deletions and point mutations upon SUP4 gene transcription. , 1982, Nucleic acids research.

[28]  P. Schimmel,et al.  Nucleotide sequence of yeast LEU2 shows 5′-noncoding region has sequences cognate to leucine , 1982, Cell.

[29]  W. Marzluff,et al.  Methylation of ribonucleic acid in a cell-free system from mouse myeloma cells. , 1982, Biochemistry.

[30]  E. Penhoet,et al.  Purification and properties of a mammalian tRNA pseudouridine synthase. , 1982, The Journal of biological chemistry.

[31]  E. D. De Robertis,et al.  Genetic analysis of the processing of a spliced tRNA. , 1982, The EMBO journal.

[32]  E. D. De Robertis,et al.  RNA processing in microinjected Xenopus oocytes. Sequential addition of base modifications in the spliced transfer RNA. , 1981, Journal of molecular biology.

[33]  R. Koski,et al.  Mutations of the yeast SUP4 tRNATyr Locus: Transcription of the mutant genes in vitro , 1980, Cell.

[34]  B. Hall,et al.  Mutations at the yeast SUP4 tRNATyr locus: DNA sequence changes in mutants lacking suppressor activity , 1980, Cell.

[35]  B. Moss,et al.  Purification and characterization of a HeLa cell transfer RNA(cytosine-5-)-methyltransferase. , 1980, The Journal of biological chemistry.

[36]  R. Fluri,et al.  The antisuppressor strain sin1 of Schizosaccharomyces pombe lacks the modification isopentenyladenosine in transfer RNA. , 1980, Journal of molecular biology.

[37]  J. Abelson,et al.  Transcription and processing of a yeast tRNA gene containing a modified intervening sequence. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[38]  R. Cortese,et al.  Order and intracellular location of the events involved in the maturation of a spliced tRNA , 1980, Nature.

[39]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[40]  J. Gorman,et al.  Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae. , 1978, Nucleic acids research.

[41]  J. Weissenbach,et al.  Pairing properties of the methylester of 5-carboxymethyl uridine in the wobble position of yeast tRNA3Arg. , 1978, Biochimica et biophysica acta.

[42]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[43]  F. Lustig,et al.  Codon-acticodon recognition in the valine codon family. , 1977, The Journal of biological chemistry.

[44]  C. Guthrie,et al.  A functional requirement for modification of the wobble nucleotide in the anticodon of a T4 suppressor tRNA , 1976, Cell.

[45]  J. Seidman,et al.  Genetic perturbations that reveal tertiary conformation of tRNA precursor molecules , 1975, Nature.

[46]  H. Ozeki,et al.  Selective modification of nucleosides of tRNA precursors accumulated in a temperature sensitive mutant of Escherichia coli , 1974, FEBS letters.

[47]  B. Barrell,et al.  Identification of tRNA precursor molecules made by phage T4. , 1973, Nature: New biology.

[48]  D. Hirsh.  Tryptophan transfer RNA as the UGA suppressor. , 1971, Journal of molecular biology.

[49]  M. Gefter,et al.  Role modifications in tyrosine transfer RNA: a modified base affecting ribosome binding. , 1969, Journal of molecular biology.

[50]  R. Rothstein.  One-step gene disruption in yeast. , 1983, Methods in enzymology.

[51]  Mathias Sprinzl,et al.  Compilation of tRNA sequences , 1983, Nucleic Acids Res..

[52]  Gerald R. Fink,et al.  Methods in Yeast Genetics: Laboratory Manual , 1981 .

[53]  F. Bolivar,et al.  Plasmids of Escherichia coli as cloning vectors. , 1979, Methods in enzymology.

[54]  Sung-Hou Kim.  Crystal Structure of Yeast tRNA phe and General Structural Features of Other tRNAs , 1979 .

[55]  H. Kang,et al.  STRUCTURE OF YEAST tRNA PRECURSORS CONTAINING INTERVENING SEQUENCES , 1979 .

[56]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .