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Biomedical subjects

E M Phizicky

Publications and source records attributed to E M Phizicky.

27 records · Page 2Linked to original sources
PubMed1990

A highly specific phosphatase from Saccharomyces cerevisiae implicated in tRNA splicing.

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.

Phosphoric Monoester Hydrolases↗
PubMed1988

Structure and function of the yeast tRNA ligase gene.

We report here the DNA sequence of the entire coding region of the Saccharomyces cerevisiae tRNA ligase gene. tRNA ligase is one of two enzymes required for tRNA splicing in yeast, and the enzyme is likely a single polypeptide with multiple activities. We find that tRNA ligase is a basic protein of 827 amino acids corresponding to a molecular weight of approximately 95,400. The inferred amino acid sequence for tRNA ligase is not significantly homologous to that of other known proteins of similar activity. In addition to the tRNA ligase reading frame and several other unidentified open reading frames, we have found two open reading frames, ORF1 and ORF2, near the 5'-end of the ligase structural gene. One of these, ORF2, produces a divergent transcript which initiates only 125 nucleotides upstream of the tRNA ligase transcript, and is present in approximately the same relative abundance as the transcript for tRNA ligase.

Amino Acid Sequence↗
PubMed1986

Saccharomyces cerevisiae tRNA ligase. Purification of the protein and isolation of the structural gene.

The tRNA ligase protein of Saccharomyces cerevisiae is one of the components required for splicing of yeast tRNA precursors in vitro. We have purified this protein to near homogeneity using an affinity elution chromatographic step. Purified tRNA ligase is a 90-kDa protein that, in addition to catalyzing the ligation of tRNA half-molecules in the coupled splicing reaction, will also ligate an artificial substrate. Using this artificial substrate, we provide evidence for the existence of a previously predicted activated intermediate in the ligation reaction. The amino acid sequence of the amino-terminal end of the protein was determined, and we have used this information to isolate the structural gene from a library of yeast DNA. We prove that this DNA encodes the tRNA ligase protein by DNA sequencing and by demonstrating overproduction of the protein.

Adenine Nucleotides↗
PubMed1981

Induction of SOS functions: regulation of proteolytic activity of E. coli RecA protein by interaction with DNA and nucleoside triphosphate.

Damage to cellular DNA or interruption of chromosomal DNA synthesis leads to induction of the SOS functions in E. coli. The immediate agent of induction is the RecA protein, which proteolytically cleaves and inactivates repressors, leading to induction of genes they control. RecA protein modified by tif mutations allows expression of SOS functions in the absence of inducing treatments. We show here that tif-mutant RecA protein is more efficient than wild-type RecA protein in interacting with DNA and nucleoside triphosphate. This result suggests that formation of a complex with DNA and nucleoside triphosphate is the critical event that activates RecA protein to destroy repressors after SOS-inducing treatments, and that damage to cellular DNA promotes this reaction by providing single-stranded DNA or active nucleoside triphosphate or both. Since dATP is the most effective nucleoside triphosphate in promoting repressor cleavage, we suggest that it is the natural cofactor of recA protein in vivo.

Adenosine Triphosphate↗
PubMedDate not supplied

A brief consideration of the SOS inducing signal.

SOS functions are induced in E. coli by treatments that damage cellular DNA or interrupt its synthesis. The biochemical basis of induction is activation of the specific proteolytic activity of recA protein, which then inactivates the lexA repressor. We discuss the development of the inducing signal in the cell.

Bacterial Proteins↗
PubMedDate not supplied

The chemical synthesis of oligoribonucleotides with selectively placed 2'-O-phosphates.

A phosphoramidite, solid support method for the chemical synthesis of oligoribonucleotides containing 2'-O-phosphate at a selected position is presented. Synthesis of these oligoribonucleotides is based on uridine- and adenosine-(2'-O-phosphate)-3'-phosphoramidites, and a new condition for removal of 2'-O-phosphate protecting groups, which does not cleave internucleotide bonds. The structure of oligoribonucleotides with 2'-O-phosphate has been proven by enzymatic digestions and dephosphorylation by yeast 2'-phosphotransferase.

Adenosine↗