title: The ability of an arginine to tryptophan substitution in Saccharomyces cerevisiae tRNA nucleotidyltransferase to alleviate a temperature-sensitive phenotype suggests a role for motif C in active site organization.
abstract: We report that the temperature-sensitive (ts) phenotype in Saccharomyces cerevisiae associated with a variant tRNA nucleotidyltransferase containing an amino acid substitution at position 189 results from a reduced ability to incorporate AMP and CMP into tRNAs. We show that this defect can be compensated for by a second-site suppressor converting residue arginine 64 to tryptophan. The R64W substitution does not alter the structure or thermal stability of the enzyme dramatically but restores catalytic activity in vitro and suppresses the ts phenotype in vivo. R64 is found in motif A known to be involved in catalysis and nucleotide triphosphate binding while E189 lies within motif C previously thought only to connect the head and neck domains of the protein. Although mutagenesis experiments indicate that residues R64 and E189 do not interact directly, our data suggest a critical role for residue E189 in enzyme structure and function. Both R64 and E189 may contribute to the organization of the catalytic domain of the enzyme. These results, along with overexpression and deletion analyses, show that the ts phenotype of cca1-E189F does not arise from thermal instability of the variant tRNA nucleotidyltransferase but instead from the inability of a partially active enzyme to support growth only at higher temperatures.
authors.affiliation: Department of Biology, Concordia University, Montréal, H4B 1R6, Canada.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Goring
authors.forename: Matthew
authors.initials: M
authors.lastname: Leibovitch
authors.forename: Ester
authors.initials: E
authors.lastname: Gea-Mallorqui
authors.forename: Shawn
authors.initials: S
authors.lastname: Karls
authors.forename: Francis
authors.initials: F
authors.lastname: Richard
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Hanic-Joyce
authors.forename: Paul B M
authors.initials: PB
authors.lastname: Joyce
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003568:Cytidine Monophosphate / Q000737:chemistry; D006358:Hot Temperature; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010641:Phenotype; D017433:Protein Structure, Secondary; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D000249:Adenosine Monophosphate; D014364:Tryptophan; D001120:Arginine; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase; D003568:Cytidine Monophosphate
keywords: Hypomorphic; Motif C; Suppressor; Temperature-sensitive; Yeast; tRNA nucleotidyltransferase