Analysis of 2'-phosphotransferase (Tpt1p) from Saccharomyces cerevisiae: evidence for a conserved two-step reaction mechanism.
Tpt1p is an essential protein responsible for the 2'-phosphotransferase step of tRNA splicing in Saccharomyces cerevisiae, in which the splice junction 2'-phosphate of ligated tRNA is transferred to NAD to form mature tRNA and ADP-ribose 1''-2'' cyclic phosphate. We showed previously that Tpt1p is a member of a family of functional 2'-phosphotransferases found in eukaryotes, eubacteria, and archaea, that the Escherichia coli protein (KptA) is highly specific for 2'-phosphorylated RNAs despite the lack of obvious natural substrates, and that KptA acts on a trinucleotide substrate through an intermediate in which RNA is ADP-ribosylated at the 2'-phosphate. This mechanism is similar to a proposed mechanism of NAD-dependent histone deacetylases. We present evidence here that this mechanism is conserved in S. cerevisiae, and we identify residues important for the second step of the reaction, during which the intermediate is resolved into products. We examined 21 Tpt1 protein variants mutated in conserved residues or blocks of residues and show that one of them, Tpt1 K69A/R71S protein, accumulates large amounts of intermediate with trinucleotide substrate due to a very slow second step. This intermediate can be trapped on beads when formed with biotin-NAD. We also show that Tpt1 K69A/R71S protein forms an intermediate with the natural ligated tRNA substrate and demonstrate that, as expected, this mutation is lethal in yeast. The high degree of conservation of these residues suggests that the entire Tpt1p family is involved in a similar two-step chemical reaction.
Steiger
Michelle A
MA
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box 712, Rochester, New York 14642, USA.
Jackman
Jane E
JE
Phizicky
Eric M
EM
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism*; D012326:RNA Splicing; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D012331:RNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D012343:RNA, Transfer; D017853:Phosphotransferases (Alcohol Group Acceptor); C106173:TPT1 protein, S cerevisiae