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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)

In a mutant strain defective in polynucleotide phosphorylase, under conditions where the enzyme becomes limiting, it is possible to demonstrate that chemical as well as functional half lives of mRNA become longer if the strain is also missing ribonuclease II. These results allow to unify in a simple model a variety of observations about turnover of RNA in a variety of bacteria.
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PMID:Polynucleotide phosphorylase can participate in decay of mRNA in Escherichia coli in the absence of ribonuclease II. 110 47

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

The Escherichia coli glyA structural gene is followed by two REP sequences and a rho-independent transcription terminator. These sequences are essential for maintaining glyA mRNA stability and gene expression by blocking the 3' to 5' exonucleolytic activities of polynucleotide phosphorylase and ribonuclease II. The results support the model of cooperative endonucleolytic and 3' to 5' exonucleolytic activities in mRNA decay.
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PMID:Escherichia coli glyA mRNA decay: the role of 3' secondary structure and the effects of the pnp and rnb mutations. 169 34

The rapid synthesis and breakdown of mRNA in prokaryotes can impose a significant energy drain on these cells. Previous in vivo studies [Duffy, J. J., Chaney, S. G. & Boyer, P. D. (1972) J. Mol. Biol. 64, 565-579; Chaney, S. G. & Boyer, P. D. (1972) J. Mol. Biol. 64, 581-591] indicated that while RNA turnover in Escherichia coli was hydrolytic, it was nonhydrolytic in Bacillus subtilis. Here we provide an explanation for these observations based on enzymatic analysis of extracts of these two organisms. RNA degradation to the mononucleotide level in E. coli extracts is due solely to two active ribonucleases, RNase II and polynucleotide phosphorylase, which act hydrolytically and phosphorolytically, respectively. RNase II activity represents close to 90% of the total activity of the extract, as expected for predominantly hydrolytic degradation in this organism. In contrast, RNase II is absent from B. subtilis extracts, and the primary mode of RNA degradation is phosphorolytic, employing the Bacillus equivalent of polynucleotide phosphorylase and releases nucleoside diphosphates as products. A low level of a Mn2(+)-stimulated, hydrolytic ribonuclease is also detectable in B. subtilis extracts. Overall, E. coli and B. subtilis extracts differ by about 20- to 100-fold, depending on the substrate, in their relative use of hydrolytic and phosphorolytic routes of RNA degradation. The relation of the mode of mRNA degradation to the environment of the cell is discussed.
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PMID:Enzymatic basis for hydrolytic versus phosphorolytic mRNA degradation in Escherichia coli and Bacillus subtilis. 170 36

We review recent evidence on the in vivo and in vitro mRNA degradation properties of 2 3'-exonucleases, ribonuclease II and polynucleotide phosphorylase. Although secondary structures in the RNA can act as protective barriers against 3' exonucleolytic degradation, it appears that this effect depends on the stability of these structures. The fact that RNase II is more sensitive to RNA secondary structure than PNPase, could account for some differences observed in messenger degradation by the 2 enzymes in vivo. Terminator stem-loop structures are often very stable and 3' exonucleolytic degradation proceeds only after they have been eliminated by an endonucleolytic cleavage. Other secondary structures preceding terminator stem-loop seem to contribute to mRNA stability against exonucleolytic decay.
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PMID:Different specificities of ribonuclease II and polynucleotide phosphorylase in 3'mRNA decay. 176 98

Two 3'-5' exoribonucleases, polynucleotide phosphorylase and ribonuclease II play a central role in the degradation of bacterial mRNA to ribonucleotides. Sequences with the potential to form stem-loop structures can stabilize upstream mRNA against 3'-5' exoribonucleolytic attack in vivo by blocking the processive activities of these enzymes. For many mRNA species stem-loop structures appear to provide a very efficient block to decay from the 3' end, such that the rate-determining step for mRNA decay occurs elsewhere in the transcript. We have examined the stalling of 3'-5' exoribonucleases at stem-loop structures in vitro. Although stem-loop structures alone can impede the progress of both enzymes, the duration of stalling at these structures in vitro is insufficient to account for the increased half-lives that they confer on mRNA in vivo. These data suggest that an additional factor, such as a stem-loop binding protein, is required for stabilization of mRNA by stem-loop structures in vivo. The implications for the regulation of mRNA stability are discussed.
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PMID:mRNA degradation by processive 3'-5' exoribonucleases in vitro and the implications for prokaryotic mRNA decay in vivo. 192 Apr 21

We review recent evidence on the in vivo and in vitro mRNA degradation properties of 2 3'-exonucleases, ribonuclease II and polynucleotide phosphorylase. Although secondary structures in the RNA can act as protective barriers against 3' exonucleolytic degradation, it appears that this effect depends on the stability of these structures. The fact that RNase II is more sensitive to RNA secondary structure than PNPase, could account for some differences observed in messenger degradation by the 2 enzymes in vivo. Terminator stem-loop structures are often very stable and 3' exonucleolytic degradation proceeds only after they have been eliminated by an endonucleolytic cleavage. Other secondary structures preceding terminator stem-loop seem to contribute to mRNA stability against exonucleolytic decay.
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PMID:Different specificities of ribonuclease II and polynucleotide phosphorylase in 3'mRNA decay. 208 42

The isolation of a temperature-sensitive allele of RNase II (rnb) by in vitro mutagenesis has permitted the demonstration that RNase II and polynucleotide phosphorylase (PNPase) are required for cell viability and mRNA turnover in Escherichia coli. Double-mutant strains carrying the pnp-7 and rnb-500 alleles (PNPase deficient and RNase II thermolabile) ceased growing in Luria broth within 30 min after shift to the nonpermissive temperature. Cessation of growth was accompanied by an accumulation of mRNA fragments 100-1500 nucleotides long. In contrast, single-mutant and wild-type control strains grew normally at the nonpermissive temperature and did not accumulate mRNA. No significant changes in rRNA patterns were observed in any of the strains.
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PMID:Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12. 241 33

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

The degradation of mRNA in Escherichia coli is thought to occur through a series of endonucleolytic and exonucleolytic steps. By constructing a series of multiple mutants containing the pnp-7 (polynucleotide phosphorylase), rnb-500 (RNase II), and ams-1 (altered message stability) alleles, it was possible to study general mRNA turnover as well as the degradation of specific mRNAs. Of most interest was the ams-1 pnp-7 rnb-500 triple mutant in which the half-life of total pulse-labeled RNA increased three- to fourfold at the nonpermissive temperature. RNA-DNA hybridization analysis of several specific mRNAs such as trxA (thioredoxin), ssb (single-stranded-DNA-binding protein), uvrD (DNA helicase II), cat (chloramphenicol acetyltransferase), nusA (N utilization substance), and pnp (polynucleotide phosphorylase) demonstrated two- to fourfold increases in their chemical half-lives. A new method for high-resolution Northern (RNA) analysis showed that the trxA and cat mRNAs are degraded into discrete fragments which are significantly stabilized only in the triple mutant. A model for mRNA turnover is discussed.
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PMID:Stabilization of discrete mRNA breakdown products in ams pnp rnb multiple mutants of Escherichia coli K-12. 245 6


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