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)

The location of poly(A) sequences in the RNA of mammalian RNA-tumor viruses was determined by enzymatic analyses. The 56-64S viral genomic RNAs, the 20-40S viral subunit RNAs, and the 4-5S poly(A) sequences excised from these viral RNAs were subjected to either hydrolysis with a 3'-OH specific exoribonuclease from Ehrlich ascites tumor cells or phosphorolysis from the 3'-termini with polynucleotide phosphorylase from Micrococcus luteus. Purified adenosine-labeled poly(A) fragments, excised from genomic viral RNAs by RNase A and T(1) digestion, were hydrolyzed with the 3'-OH specific exoribonuclease for various periods of time. Poly(U) filter binding studies of the residual poly(A) indicated that 97% of the poly(A) fragments were hydrolyzed. Adenosine-labeled genomic and subunit viral RNAs and excised poly(A) fragments were phosphorolyzed from their 3'-termini for various periods of time with polynucleotide phosphorylase. The degree of phosphorolysis was monitored by poly(U) filter binding studies, and CCl(3)COOH insolubility and solubility determinations. There was an initial preferential rate of phosphorolysis of the poly(A) sequences of genomic and subunit viral RNAs as compared to the total adenosine-labeled viral RNAs. The data from these two different enzymatic mechanisms of action indicated conclusively that the poly(A) sequences were located at the 3'-termini of genomic and subunit viral RNAs.
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PMID:Polyriboadenylate sequences at the 3'-termini of ribonucleic acid obtained from mammalian leukemia and sarcoma viruses. 437 12

Three polynucleotide phosphorylase mutations, isolated in heavily mutagenized Escherichia coli strains Q7, Q13, and Q27, were characterized after their transfer by P1 transduction to nearly isogenic strains which lack ribonuclease I. Each strain has a different altered form of polynucleotide phosphorylase. One enzyme exhibited sharply reduced activity under all conditions tested. A second had reduced activity which was stimulated by Mn(++). The third enzyme was thermolabile and could be >95% inactivated in vivo at 44 C and pH 6 if the cells were prevented from growing; during growth under these and other conditions, the full enzyme level was maintained. The strains showed no differences from the wild type in their growth rates, their adjustments to changes in media and temperature, or their recoveries from starvation.
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PMID:Characterization of polynucleotide phosphorylase mutants of Escherichia coli. 488 20

A new route for the synthesis of 1-(beta-D-allofuranosyl)uracil ("allo-uridine") and the corresponding 6'-deoxy-derivative ("6'-deoxy-allo-uridine") as well as for 1-(beta-D-altrofuranosyl) uracil ("altro-uridine") is described. NMR studies of allo-uridine revealed a preferred conformation with the base in anti-position, C-2'-endo-pucker of the sugar moiety, the 5'-OH-group above the furanose ring and the 5'-CH2OH-group in a gt position with the OH-group in the plane of the furanose ring. The same conformation is found for the 5'- and 6'-phosphate, indicated by the influence of the phosphate group on the H-6 signal. Allo-uridine is phosphorylated by the phosphotransferases from carrot and from malt sprouts only in the 6'-position. The phosphate ester is hydrolysed by unspecific phosphatases but not by 5'-nucleotidase. A (3' leads to 6')-dinucleoside phosphate is formed by pancreatic ribonuclease with 2',3'-cyclic cytidylic acid and allo-uridine. It is split by nuclease S1, but not by snake-venom phosphodiesterase. It has no primer activity for polynucleotide phosphorylase. All-uridine 6'-diphosphate could not be prepared enzymatically by nucleotide kinase or by chemical methods, where 5',6'-cyclic phosphates are formed, which are hydrolysed exclusively to 6'-monophosphates.
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PMID:Synthesis, conformation and enzymatic properties of 1-(beta-D-allofuranosyl)uracil and some derivatives. 631 65

We report the polymerization of 5-mercuriuridine-5'-diphosphate (ppUHgX), in the presence of an excess of beta-mercaptoethanol, with polynucleotide phosphorylase from E. coli. A degradation of mercurated nucleotides with mercaptans was observed and about 30% of incorporation of mercury in the polymer was obtained after treatment with pancreatic RNase. The influence of ppUHgX and pUHgX with or without beta-mercaptoethanol was also studied on the polymerization of UDP. The ppUHgX did not polymerize in the absence of beta-mercaptoethanol.
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PMID:Enzymatic polymerization of 5-mercuriuridine-5'-diphosphate with polynucleotide phosphorylase from E. coli. 639 3

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

Although polyadenylation has commonly been regarded as a special feature of eukaryotic messenger RNA, there are many reports of polyA tails on bacterial RNA (for example, refs 3-8). In Escherichia coli, adenylation mediated by the pcnB gene greatly accelerates decay of RNA I, an antisense repressor of replication of ColE1 type plasmids that resembles highly structured transfer RNA but shows the rapid turnover characteristic of mRNA. Here we report that both 3' adenylation and 5' phosphorylation affect the rate of digestion of RNA I by the 3' exonuclease, polynucleotide phosphorylase; conversely, mutation of the polynucleotide phosphorylase-encoding pnp gene affects ribonuclease acting at the 5' end. Together these findings indicate that enzymes attacking RNA I at its separate termini can interact functionally. Additionally, our discovery that adenylation-mediated degradation by polynucleotide phosphorylase imparts an mRNA-like half-like to RNA I suggests a possible mechanism to account for the rapid decay of mRNA in E. coli.
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PMID:RNA degradation in Escherichia coli regulated by 3' adenylation and 5' phosphorylation. 753 64

As part of our genetic analysis of mRNA decay in Escherichia coli K-12, we examined the effect of the pcnB gene [encoding poly(A) polymerase I] on message stability. Eliminating poly(A) polymerase I (delta pcnB) dramatically stabilized the lpp, ompA, and trxA transcripts. The half-lives of individual mRNAs were increased in both a delta pcnB single mutant and a delta pcnB pnp-7 rnb-500 rne-1 multiple mutant. We also found mRNA decay intermediates in delta pcnB mutants that were not detected in control strains. By end-labeling total E. coli RNA with [32P]pCp and T4 RNA ligase and then digesting the RNA with RNase A and T1, we showed that many RNAs in a wild-type strain contained poly(A) tails ranging from 10 nt to > 50 nt long. When polynucleotide phosphorylase, RNase II, and RNase E were absent, the length (> 100 nt) and number (10- to 20-fold) of the poly(A) tails increased. After transcription initiation was stopped with rifampicin, polyadenylylation apparently continued. Deleting the structural gene for poly(A) polymerase I (pcnB) reduced the amount of 3'-terminal poly(A) sequences by > 90%. We propose a model for the role of polyadenylylation in mRNA decay.
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PMID:Polyadenylylation helps regulate mRNA decay in Escherichia coli. 789 80

Hammerhead ribozymes are small catalytic RNA molecules that can be designed to specifically cleave other RNAs. These ribozymes have exhibited low efficiency when examined inside cells, perhaps in part because of their sensitivity to intracellular RNases. In an effort to better understand intracellular degradation of small, foreign RNAs and to develop more stable ribozymes, the ability of Escherichia coli RNase mutants to digest ribozymes was examined. In soluble extracts, most (80 to 90%) of the endonucleolytic activity was due to RNases I and I*, since degradative activity was inhibited by Mg2+ and by the rna-2 mutation. Degradation by exonucleolytic activities was temperature sensitive in extracts from an rna pnp rnb(Ts) triple mutant but not in extracts from an rna rnb(Ts) double mutant. Thus, the products of rnb and pnp, RNase II and polynucleotide phosphorylase, respectively, appear to be the major exonucleases that degrade hammerhead ribozymes. Examination of intracellular degradation revealed that RNases I and I* contributed to about half of the degradative activity as judged by comparison of the rate of ribozyme decay in wild-type and rna-2 mutant cells. Little additional effect was observed in rne(RNase E) and rnc (RNaseIII) mutants. Taken together, these data indicate that hammerhead ribozymes are digested largely by the degradative class of RNase (RNases I, I* and II and polynucleotide phosphorylase).
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PMID:RNases involved in ribozyme degradation in Escherichia coli. 862 92

In the presence of Mg2+ ions, polynucleotide phosphorylase (PNPase, EC 2.7.7.8) is known to synthesize RNA-like polymers using ribonucleoside-5'-diphosphate (NDP) substrates but to be unable to utilize deoxyribonucleoside substrates. Our experiments show that when MgCl2 is replaced by FeCl3, PNPase becomes able to synthesize deoxyheteropolymers using deoxyribonucleoside-5'-diphosphates (dNDPs). The deoxyheteropolymer formed from the four dNDPs is degraded by pancreatic DNase, but not by RNase, and is readily used as a template by DNA-dependent DNA polymerase. Synthesis of this DNA-like polymer is accomplished de novo without the help of any primer or preexisting template. What is more, dA/dG and dC/dT ratios of polymers synthesized by different bacterial PNPases closely match ratios found in DNA of the bacterial species the enzyme came from.
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PMID:De Novo Synthesis of DNA-Like Molecules by Polynucleotide Phosphorylase In Vitro 866 1

In Escherichia coli, ribonuclease E (RNase E) is a key endonuclease in mRNA decay. We have analysed the role of E coli RNase E on the degradation of a heterologous cytochrome c3 (cyc) mRNA from Desulfovibrio vulgaris Hildenborough. The decay of the cyc transcript in wild-type and mutant E coli cells was followed and the degradation intermediates analysed by Northern blotting and S1 protection analysis. The half-life of total cyc mRNA intermediates was increased in the RNase E mutant. A number of degradation intermediates were stabilised, and new species arose. However, some species decayed faster in the met5 mutant at the non-permissive temperature, suggesting that RNase E might inhibit their degradation. The results indicate that RNase E is involved in cyc mRNA degradation, and, interestingly, decay of certain intermediates could be reduced by this enzyme activity. This may suggest a functional interaction between RNase E and exonucleases, like polynucleotide phosphorylase.
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PMID:RNase E can inhibit the decay of some degradation intermediates: degradation of Desulfovibrio vulgaris cytochrome c3 mRNA in E coli. 887 97


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