ABSTRACT
For the identification of yeast genes specifying biochemical activities, a genomic strategy that is rapid, sensitive, and widely applicable was developed with an array of 6144 individual yeast strains, each containing a different yeast open reading frame (ORF) fused to glutathione S-transferase (GST). For the identification of ORF-associated activities, strains were grown in defined pools, and GST-ORFs were purified. Then, pools were assayed for activities, and active pools were deconvoluted to identify the source strains. Three previously unknown ORF-associated activities were identified with this strategy: a cyclic phosphodiesterase that acts on adenosine diphosphate-ribose 1"-2" cyclic phosphate (Appr>p), an Appr-1"-p-processing activity, and a cytochrome c methyltransferase.
Subject(s)
Fungal Proteins/genetics , Genes, Fungal , Genetic Techniques , Open Reading Frames , Saccharomyces cerevisiae/genetics , Adenosine Diphosphate Ribose/analogs & derivatives , Adenosine Diphosphate Ribose/metabolism , Fungal Proteins/metabolism , Glutathione Transferase/genetics , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/isolation & purification , Histone-Lysine N-Methyltransferase/metabolism , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/isolation & purification , Phosphoric Diester Hydrolases/metabolism , Recombinant Fusion Proteins/isolation & purification , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
Gene Expression Regulation, Fungal , Genes, Fungal , Phosphotransferases (Alcohol Group Acceptor)/genetics , RNA Splicing/genetics , RNA, Fungal/genetics , RNA, Transfer/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Molecular Sequence Data , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Sequence AlignmentABSTRACT
Transfer RNA (tRNA) splicing is essential in Saccharomyces cerevisiae as well as in humans, and many of its features are the same in both. In yeast, the final step of this process is removal of the 2' phosphate generated at the splice junction during ligation. A nicotinamide adenine dinucleotide (NAD)-dependent phosphotransferase catalyzes removal of the 2' phosphate and produces a small molecule. It is shown here that this small molecule is an NAD derivative: adenosine diphosphate (ADP)-ribose 1"-2" cyclic phosphate. Evidence is also presented that this molecule is produced in Xenopus laevis oocytes as a result of dephosphorylation of ligated tRNA.
Subject(s)
Adenosine Diphosphate Ribose/analogs & derivatives , RNA Splicing , RNA, Fungal/metabolism , RNA, Transfer/metabolism , Saccharomyces cerevisiae/genetics , Adenosine Diphosphate Ribose/chemistry , Adenosine Diphosphate Ribose/metabolism , Animals , Cyclic ADP-Ribose , Endoribonucleases/metabolism , NAD/chemistry , NAD/metabolism , Oocytes/metabolism , Phosphates/metabolism , Phosphorylation , Phosphotransferases/metabolism , XenopusABSTRACT
An enzyme from Saccharomyces cerevisiae which removes the splice junction 2'-phosphate from ligated tRNA appears to require NAD+. This two-component enzyme has been previously implicated in tRNA splicing because of its specificity for substrates bearing an internal 2'-phosphate and because of the absence of other observed proteins that can efficiently catalyze the same activity after fractionation of the extracts. We show here that component I of this enzyme is heat-stable, chromatographs as a small molecule, can be substituted efficiently by NAD+, and comigrates with NAD+ on a reversed-phase column. Dephosphorylation of ligated tRNA in the presence of component I or NAD+ is accompanied by stoichiometric transfer of the splice junction 2'-phosphate to an unidentified acceptor molecule.
Subject(s)
NAD/metabolism , Organophosphorus Compounds/metabolism , RNA Splicing , RNA, Transfer/metabolism , Saccharomyces cerevisiae/enzymology , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Phosphorylation , Substrate SpecificityABSTRACT
We identified and partially purified a phosphatase from crude extracts of Saccharomyces cerevisiae cells that can catalyze the last step of tRNA splicing in vitro. This phosphatase can remove the 2'-phosphate left over at the splice junction after endonuclease has removed the intron and ligase has joined together the two half-molecules. We suggest that this phosphatase is responsible for the completion of tRNA splicing in vivo, based primarily on its specificity for the 2'-phosphate of spliced tRNA and on the resistance of the splice junction 2'-phosphate to a nonspecific phosphatase. Removal of the splice junction 2'-phosphate from the residue adjacent to the anticodon is likely necessary for efficient expression of spliced tRNA. The phosphatase appears to be composed of at least two components which, together with endonuclease and ligase, can be used to reconstitute the entire tRNA-splicing reaction.
