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 oligoribonucleotide, A-A-A-C-U-U-U-Gp, constituting a segment of RNA bacteriophage Qbeta coat protein gene was efficiently synthesized at a milligram scale by a combination of enzymatic methods using bacteriophage T4 RNA ligase and the thermophilic polynucleotide phosphorylase. A-A-A-Cp was synthesized from A-A-A and pCp by the newly developed mononucleotide addition method using T4 RNA ligase in a yield of 83%, followed by dephosphorylation with bacterial alkaline phosphatase to obtain A-A-A-C. pU-U-U-Gp was synthesized from pU-U-U and GDP by the simultaneous action of polynucleotide phosphorylase and RNase T1 in a yield of 32%. finally, the two oligonucleotides (A-A-A-C and pU-U-U-Gp) were ligated with T4 RNA ligase and the octanucleotide, A-A-A-C-U-U-U-Gp, was obtained in a yield of 85%.
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PMID:Enzymatic synthesis of a segment of bacteriophage Qbeta coat protein gene. 41 26

Under the conditions that RNA ligase converts the tetranucleotide, pA-A2-A, to larger polynucleotides, no such polymerization can be detected with the derivative, pA-A2-A(MeOEt), that possesses a terminal 2'-0-(alpha-methoxyethyl) group. The protection against self condensation offered by the methoxyethyl group in this system allows the specific joining of donor and acceptor oligonucleotides in reaction mixtures containing equimolar concentrations of the two species. Thus, the enzyme, together with ATP, converts equimolar quantities of A-A2-A and pA-A2-A(MeOEt) to A-A6-A(MeOEt) in 55% yield, while a similar reaction with A-A2-A and pU-U2-U(MeOEt) results in a 40% yield of A-A3-U3-U(MeOEt). The intermediate in these ligations is a disubstituted pyrophosphate composed of the donor molecule and the adenylate moiety deriving from ATP. In the case of the intermediate arising from the blocked adenosine tetranucleotide, the assigned structure, A5'pp5'A-A2-A(MeOEt), has been confirmed by chemical synthesis. The pyrophosphate derivative is able to participate in joining reactions in the absence of ATP. These observations constitute an efficient approach to the synthesis of larger polynucleotides from a specific series of oligonucleotide blocks since (i), the methoxyethyl group can be easily introduced into each oligonucleotide using the single addition reaction catalyzed by polynucleotide phosphorylase in the presence of a 2'-0-(alpha-methoxyethyl)nucleoside 5'-diphosphate, and (ii), the blocking group may be readily removed under mild conditions after each successive ligation reaction. Two other octanucleotides, I-I2-A-U3-U and U-U2-C-I3-A, have also been synthesized by this method, and these molecules correspond (with I substituting for G) to sequences appearing near the 3' terminus of the 6S RNA transcribed from phage lambda DNA. The terminal 3'-phosphate group serves equally well as a blocking group for specific ligation reactions in that the ligase converts equimolar amounts of A-A2-A and pA-A2-Ap to A-A6-Ap in 50% yield.
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PMID:The use of terminal blocking groups for the specific joining of oligonucleotides in RNA ligase reactions containing equimolar concentrations of acceptor and donor molecules. 100 14

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

In the present paper the results of enzymatic synthesis of yeast tRNA1Val fragments have been summarized. It is shown that complex use of nucleolytic enzymes is a convenient and effective method of synthesis of the defined sequence oligoribonucleotides. The consecutive use of different nucleolytic enzymes (ribonucleases with different substrate specificity and polynucleotide phosphorylase) and RNA ligase has permitted to obtain various fragments (or their analogs) of T psi-loop, D-arm, anticodon arm and acceptor stem. Some fragments containing modified nucleosides such as tetranucleotide GpDpCpGp (fragment 15-18), octanucleotide GpUpCpUpApGpDpC (analog of fragment 10-17), nonanucleotide GpTpUpCpGpApUpCpC (analog of T psi-loop), decanucleotide psi pCpUpGpCpUpUpIpApC (analog of fragment 27-36), hexanucleotide CpApCpGpCpA (fragment 36-41) and others were synthesized.
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PMID:[Enzymatic synthesis of tRNA fragments]. 639 Jan 71

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

We have examined the expression of pnp encoding the 3'-5'-exoribonuclease, polynucleotide phosphorylase, in Streptomyces antibioticus. We show that the rpsO-pnp operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both pnp and the gene for ribosomal protein S15 (rpsO) and a second, Ppnp, located in the rpsO-pnp intergenic region. Unlike the situation in Escherichia coli, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type S. antibioticus mycelia. The Ppnp transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5' ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The Ppnp sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in rspO-pnp structure and function between S. antibioticus and E. coli. In E. coli, pnp autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an rpsO-pnp intergenic hairpin. Computer modeling of the secondary structure of the S. antibioticus readthrough transcript predicts a stem-loop structure analogous to that in E. coli. However, our analysis suggests that while the readthrough transcript observed in S. antibioticus may be processed by an RNase III-like activity, transcripts originating from Ppnp are not. Furthermore, the S. antibioticus rpsO-pnp intergenic region contains two open reading frames. The larger of these, orfA, may be a pseudogene. The smaller open reading frame, orfX, also observed in Streptomyces coelicolor and Streptomyces avermitilis, may be translationally coupled to pnp and the gene downstream from pnp, a putative protease.
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PMID:Organization and expression of the polynucleotide phosphorylase gene (pnp) of Streptomyces: Processing of pnp transcripts in Streptomyces antibioticus. 1512 78