RESUMO
tRNA splicing in the yeast Saccharomyces cerevisiae requires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2'-phosphotransferase to transfer the splice junction 2'-phosphate from ligated tRNA to NAD, producing ADP ribose 1"-2" cyclic phosphate (Appr>p). We show here that functional 2'-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicans and Schizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules. Surprisingly, functional 2'-phosphotransferase is found also in the bacterium Escherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2'-phosphate. Analysis of the database shows that likely members of the 2'-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidus and Pyrococcus horikoshii). Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2'-phosphotransferase into Eubacteria, suggesting that the 2'-phosphotransferase has been present there since close to the time that the three kingdoms diverged. Although 2'-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential in E. coli, the continued presence of 2'-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein.
Assuntos
Endorribonucleases/genética , Escherichia coli/enzimologia , Splicing de RNA , RNA de Transferência/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Arabidopsis/enzimologia , Candida albicans/enzimologia , Endorribonucleases/química , Endorribonucleases/metabolismo , Escherichia coli/genética , Biblioteca Gênica , Humanos , Mamíferos , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Schizosaccharomyces/enzimologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
tRNA splicing is essential in yeast and humans and presumably all eukaryotes. The first two steps of yeast tRNA splicing, excision of the intron by endonuclease and joining of the exons by tRNA ligase, leave a splice junction bearing a 2'-phosphate. Biochemical analysis suggests that removal of this phosphate in yeast is catalyzed by a highly specific 2'-phosphotransferase that transfers the phosphate to NAD to form ADP-ribose 1"-2" cyclic phosphate. 2'-Phosphotransferase catalytic activity is encoded by a single essential gene, TPT1, in the yeast Saccharomyces cerevisiae. We show here that Tpt1 protein is responsible for the dephosphorylation step of tRNA splicing in vivo because, during nonpermissive growth, conditional lethal tpt1 mutants accumulate 2'-phosphorylated tRNAs from eight different tRNA species that are known to be spliced. We show also that several of these tRNAs are undermodified at the splice junction residue, which is always located at the hypermodified position one base 3' of the anticodon. This result is consistent with previous results indicating that modification of the hypermodified position occurs after intron excision in the tRNA processing pathway, and implies that modification normally follows the dephosphorylation step of tRNA splicing in vivo.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool) , Splicing de RNA , RNA Fúngico , RNA de Transferência , Proteínas de Saccharomyces cerevisiae , Genes Fúngicos , Genes Letais , Modelos Químicos , Mutação/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Ribonucleotídeos/análise , Splicing de RNA/genética , RNA Fúngico/química , RNA Fúngico/genética , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência de Leucina/química , RNA de Transferência de Tirosina/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
The last step of tRNA splicing in the yeast Saccharomyces cerevisiae is catalyzed by an NAD-dependent 2'-phosphotransferase, which transfers the splice junction 2'-phosphate from ligated tRNA to NAD to produce ADP-ribose 1"-2" cyclic phosphate. We have purified the phosphotransferase about 28,000-fold from yeast extracts and cloned its structural gene by reverse genetics. Expression of this gene (TPT1) in yeast or in Escherichia coli results in overproduction of 2'-phosphotransferase activity in extracts. Tpt1 protein is essential for vegetative growth in yeast, as demonstrated by gene disruption experiments. No obvious binding motifs are found within the protein. Several candidate homologs in other organisms are identified by searches of the data base, the strongest of which is in Schizosaccharomyces pombe.
Assuntos
Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Splicing de RNA/genética , RNA Fúngico/genética , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Alinhamento de SequênciaRESUMO
Adenosine diphosphate (ADP)-ribose 1",2"-cyclic phosphate (Appr > p) is produced as a result of transfer RNA (tRNA) splicing in the yeast Saccharomyces cerevisiae and probably in other eukaryotes. Endonucleolytic cleavage and ligation result in a mature length tRNA with a 2'-phosphate at the splice junction. This 2'-phosphate is transferred to NAD to produce Appr > p. Metabolism of Appr > p requires hydrolysis of the 1",2"-cyclic phosphate linkage. We show here that yeast has a unique cyclic phosphodiesterase that can hydrolyze Appr > p, ribose 1,2-cyclic phosphate, and ribose 1,3-cyclic phosphate to the corresponding ribose 1-phosphate derivatives. The cyclic phosphodiesterase is highly specific for Appr > p; there is 20-fold less activity on ribose 1,3-cyclic phosphate and no detectable activity on nucleoside 2',3'-cyclic phosphates. A similar cyclic phosphodiesterase is present in wheat germ. The wheat germ cyclic phosphodiesterase activity co-chromatographs with a 2',3'-cyclic nucleotide 3'-phosphodiesterase that was previously identified and purified. The purified wheat germ enzyme has a distinct preference for Appr > p and ribose cyclic phosphate compared to guanosine 2',3'-cyclic phosphate and shares other biochemical characteristics with the yeast enzyme.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Splicing de RNA , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Triticum/genéticaRESUMO
We show here that yeast tRNA ligase protein is essential in the cell and participates in joining together tRNA half-molecules resulting from excision of the intron by the splicing endonuclease. A haploid yeast strain carrying a chromosomal deletion of the ligase gene is viable only if ligase protein can be supplied from a plasmid copy of the gene. When synthesis of the plasmid-borne ligase gene is repressed, cells eventually die and accumulate endonuclease cut but unligated half-molecules and intervening sequences. Half-molecules that accumulate appear to be fully end-processed. Two temperature-sensitive ligase mutant strains have been isolated; these strains accumulate a similar set of unligated half-molecules at the nonpermissive temperature.
