Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.8 (polynucleotide phosphorylase)
723 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro. To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains. Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase. Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability. The rph mutation also had dramatic effects on tRNA metabolism. Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases. Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence. These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors.
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PMID:RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells. 164 89

Escherichia coli RNase PH is a phosphate-dependent exoribonuclease that has been implicated in the 3' processing of tRNA precursors. It degrades RNA chains in a phosphorolytic manner releasing nucleoside diphosphates as products. Here we show that RNase PH also catalyzes a synthetic reaction, the addition of nucleotides to the 3' termini of RNA molecules. The synthetic activity co-purifies with RNase PH throughout an extensive enrichment indicating that it is due to the same enzyme. The synthetic activity can incorporate all nucleoside diphosphates, but not triphosphates, and is strongly inhibited by Pi, but not PPi. Various RNA molecules stimulate nucleotide incorporation, and with tRNA the 3' end of the molecule serves a primer function. RNA chains as long as 40 residues can be synthesized in this system. As with polynucleotide phosphorylase, the synthetic activity of RNase PH apparently represents the reversal of the degradative reaction.
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PMID:RNase PH catalyzes a synthetic reaction, the addition of nucleotides to the 3' end of RNA. 170 83

A polynucleotide phosphorylase was isolated from the Thermus thermophilus protein fractions, obtained at different steps of purification of elongation factors, and immobilized on agarose activated with cyanogen bromide and macroporous glass modified with (3,3-diethoxypropyl)triethoxysilane. The preparations of the native and immobilized enzyme catalyzed rather efficiently the addition of adenylyl and guanylyl residues to oligonucleotide primers, in contrast to the E. coli and M. luteus polynucleotide phosphorylases. Tri-, tetra- and pentanucleotides with 3'-terminal guanosine and adenosine were obtained including structural analogues of the anticodon fragment 34-37 of yeast tRNA(Phe).
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PMID:[Stepwise synthesis of oligonucleotides. XXXV. Native and immobilized polynucleotide phosphorylase from Thermus thermophilus in oligoribonucleotide synthesis]. 240 Apr 8

Final trimming of the 3' terminus of tRNA precursors in Escherichia coli is thought to proceed by an exonucleolytic mechanism. However, mutant strains lacking as many as four exoribonucleases known to act on tRNA still grow normally and process tRNA normally. Extracts from such a multiple-RNase-deficient strain accurately mature tRNA precursors exonucleolytically in vitro in a reaction that requires inorganic phosphate. Here we show that this reaction is not due to polynucleotide phosphorylase (PNPase) but, rather, that it is mediated by a phosphate-requiring exonuclease that we have named RNase PH. Purified PNPase is incapable of completely processing tRNA precursors, and extracts from a PNPase- strain retain full activity for phosphorolytic processing. Although both PNPase and RNase PH act in a phosphorolytic manner, they differ substantially in size and substrate specificity. RNase PH has a molecular mass of 45-50 kDa and favors tRNA precursors as substrates. The possible physiological role of RNase PH and the advantages of phosphorolytic processing are discussed.
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PMID:RNase PH: an Escherichia coli phosphate-dependent nuclease distinct from polynucleotide phosphorylase. 245 97

Oligouridylates of varying chain lengths were synthesized by polynucleotide phosphorylase and cyclized by RNA ligase. Over chain lengths from 7 to 15, the bindings of the cyclized and linear oligomers to polyadenylate were measured on the basis of differential migration of bound and free oligomers on a gel exclusion column. Binding of the cyclized oligomers was found to be far weaker than that of their linear counterparts of equal length. Such a general reduction in base-pairing capacity due to the cyclized conformation, by limiting the strength of unintended base-pairing without obstructing the possible development of strong specific base-pairing, may represent an advantage important to the function and evolution of loop structures in tRNA and other RNA molecules.
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PMID:Complementary base-pairing properties of cyclized and linear oligonucleotides. 359 58

The preparation of phenylalanyl-tRNA terminating in 3'-deoxyadenosine has been achieved by incubation of abbreviated tRNA (tRNA-CpC(OH)) with 3'-deoxyadenosine 5'-diphosphate and polynucleotide phosphorylase (EC 2.7.7.8), followed by aminoacylation. The isomeric phenylalanyl-tRNA terminating in 2'-deoxyadenosine was constructed by incubation of tRNA-CpC(OH) with 2'-deoxy-3'-O-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase. While tRNA is aminoacylated at the 2'-position, only the 3'-aminoacyl-tRNA is active as a peptide acceptor in the peptidyltransferase reaction. Both modified tRNAs were bound to the A-site as efficiently as unmodified tRNA, but neither was so efficient at P-site binding or as an acceptor in the peptidyltransferase reaction. Neither of the modified tRNAs acted as a donor in the peptidyltransferase reaction.
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PMID:Isomeric phenylalanyl-tRNAs. Position of the aminoacyl moiety during protein biosynthesis. 461 16

We have used an in vitro Escherichia coli tRNA processing system to investigate the specific role of individual exoribonucleases in the 3' maturation of tRNA precursors. The processing of pre-tRNA(Tyr)su3+ and pre-tRNA(2Arg) was studied using extracts from cells lacking one or multiple exoribonucleases or using purified RNases. Earlier genetic studies had suggested that multiple exoribonucleases contributed to the maturation of tRNA precursors, and this was proven directly in the studies described here. Complete 3' processing required the combined action of multiple exoribonucleases, and each RNase showed distinct specificities for maturation of the different parts of the 3' precursor segment. RNase II and polynucleotide phosphorylase were most effective in shortening long 3' trailer sequences to intermediates with 2-4 extra 3' residues. Final trimming of the last few 3' nucleotides of these precursors was carried out most efficiently by RNases T and PH, but the two enzymes differed in their specificity for individual nucleotide positions. Depending on the tRNA precursor, the relative importance of the various RNases to the overall maturation process differed. We also showed that purified exoribonucleases can completely complement mutant extracts and that tRNA maturation can be totally reconstructed in vitro using purified enzymes. These studies provide the first detailed information about the specific role of individual exoribonucleases in tRNA processing, and bring us closer to defining a complete E. coli tRNA maturation pathway.
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PMID:The role of individual exoribonucleases in processing at the 3' end of Escherichia coli tRNA precursors. 750 97

A second poly(A) polymerase (PAP II) has been identified in Escherichia coli using a strain carrying a deletion of pcnB (the structural gene for PAP I; Cao and Sarkar, 1992b) and pnp-7 (a null mutation in the structural gene for polynucleotide phosphorylase). While PAP I has a M(r) of 53,000, PAP II is a smaller protein with a native M(r)-35,000. PAP II differs from PAP I in preferring poly(A) over tRNA primers and being more thermolabile. The presence of multiple poly(A) polymerases in E. coli raises interesting questions regarding the role of polyadenylation in mRNA synthesis and decay.
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PMID:Identification of a second poly(A) polymerase in Escherichia coli. 790 63

We report the cloning and characterization of a cell division gene, herein designated divIC, from the gram-positive, spore-forming bacterium Bacillus subtilis. This gene was previously identified on the basis of a temperature-sensitive mutation, div-355, that blocks septum formation at restrictive temperatures. We show that the divIC gene is a 125-codon open reading frame that is capable of encoding a protein of 14.7 kDa and that div-355 is a 5-bp duplication near the 3' end of the open reading frame. We also show that divIC is an essential gene by use of an in vitro-constructed null mutation. In confirmation and extension of earlier results, we show that divIC is necessary for both vegetative and sporulation septum formation, and we demonstrate that it is required for the activation of genes expressed under the control of the sporulation transcription factors sigma F and sigma E. The divIC gene is located 1.3 kb upstream of the coding sequence for the sporulation gene spoIIE. Between divIC and spoIIE is a 128-codon open reading frame whose predicted product contains a region of similarity to the RNA-binding domains of polynucleotide phosphorylase and ribosomal protein S1 from Escherichia coli and two putative tRNA genes for methionyl-tRNA and glutamyl-tRNA, the gene order being divIC orf128 tRNA(Met) tRNA(Glu) spoIIE.
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PMID:Characterization of a cell division gene from Bacillus subtilis that is required for vegetative and sporulation septum formation. 811 87

Escherichia coli cells lacking both polynucleotide phosphorylase (PNPase) and RNase PH, the only known P(i)-dependent exoribonucleases, were previously shown to grow slowly at 37 degrees C and to display a dramatically reduced level of tRNA(Tyr)su3+ suppressor activity. Here we show that the RNase PH-negative, PNP-negative double-mutant strain actually displays a reversible cold-sensitive phenotype and that tRNA biosynthesis is normal. In contrast, ribosome structure and function are severely affected, particularly at lower temperatures. At 31 degrees C, the amount of 50S subunit is dramatically reduced and 23S rRNA is degraded. Moreover, cells that had been incubated at 42 degrees C immediately cease growing and synthesizing protein upon a shift to 31 degrees C, suggesting that the ribosomes synthesized at the higher temperature are defective and unable to function at the lower temperature. These data indicate that RNase PH and PNPase play an essential role that affects ribosome metabolism and that this function cannot be taken over by any of the hydrolytic exoribonucleases present in the cell.
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PMID:An essential function for the phosphate-dependent exoribonucleases RNase PH and polynucleotide phosphorylase. 920 58


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