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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 genes encoding ribosomal protein S15 (rpsO) and polynucleotide phosphorylase (pnp) occupy adjacent positions and are oriented in the same direction on the Escherichia coli chromosomes. The nucleotide sequence of the region controlling the expression of these two genes has been determined. Two in-phase gene fusions between pnp and lacZ were constructed. The fusions define the translational reading frame of the pnp gene and indicate that the expression of pnp is independent of the upstream rpsO gene. Transcript mapping with nuclease S1 demonstrated that the two genes are transcribed from separate promoters and that the rpsO-pnp intergenic space contains a strong transcriptional terminator. The transcriptional start points have been localized.
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PMID:Promoter activity and transcript mapping in the regulatory region for genes encoding ribosomal protein S15 and polynucleotide phosphorylase of Escherichia coli. 300 22

The rpsO gene of Escherichia coli, which encodes ribosomal protein S15 is located at 69 minutes on the chromosome. It is adjacent to the pnp gene, which encodes polynucleotide phosphorylase. The two genes are separated by 249 nucleotides and are transcribed in the same direction. We report here in vivo S1 nuclease mapping and in vitro transcription experiments that demonstrate that rpsO and pnp are cotranscribed from a promoter P1, located 108 nucleotides upstream from rpsO, and that another promoter P2, located between the two genes 158 nucleotides upstream from pnp, also directs the transcription of pnp. Transcription from P1 can either terminate at the terminator t1 identified in vivo and in vitro, 18 nucleotides downstream from rpsO, or transcribe through t1 and into pnp. Comparison of the transcripts synthesized in wild-type and RNase III-deficient strains of E. coli shows that all the P1 readthrough transcripts and P2 transcripts are cleaved by RNase III. Two specific cuts are made by RNase III in a double-stranded structure about 100 nucleotides upstream rpsO. We also found that some transcripts of this operon start 47 nucleotides downstream from rpsO, in the region of t1. No promoter has been identified in this region. This mRNA is attributed to an endonucleolytic cleavage of the polycistronic transcripts and the location of the cut is named M. The order of the transcription signals and of the maturation sites in relation to rpsO and pnp can be summarized as follows: P1, rpsO, t1, M, P2, RNase III-processing sites, pnp. The possible roles of mRNA processing events in the expression of rpsO-pnp operon are discussed.
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PMID:Initiation, attenuation and RNase III processing of transcripts from the Escherichia coli operon encoding ribosomal protein S15 and polynucleotide phosphorylase. 300 65

Poly(7-deazaguanylic acid) was enzymatically synthesized by the polymerization of 7-deazaguanosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield. The homopolymer showed a similar thermal and total hypochromicity to poly(G) at the long wavelength absorption maximum. No sigmoid melting profile was observed for poly(c7G) as is found for poly(G), implying a single-stranded structure in aqueous solution. From the circular dichroism spectra it can be concluded that the 7-deazapurine nucleotide is much more flexible than the purine nucleotide. In analogy to poly(G), the homopolymer poly(c7G) forms a 1:1 complex with poly(C) under neutral conditions, melting at a similar temperature to the poly(G) complex. However, at pH 2.5, where a poly(G) X 2poly(C) complex is observed, poly(c7G) still binds only one poly(C) strand. This is due to the lack of N-7 in poly(c7G), not allowing Hoogsteen base pair formation, which occurs with poly(G). RNase T1 cleaves poly(c7G), indicating that N-7 of guanosine is not a requirement for nucleotide binding to the enzyme, as has been suggested. Because of the single-stranded structure of poly(c7G), the polynucleotide chain is rapidly hydrolyzed by the single-strand-specific nuclease S1, whereas multistranded poly(G) is completely resistant.
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PMID:Poly(7-deazaguanylic acid), the homopolynucleotide of the parent nucleoside of queuosine. 628 79

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

Poly(2-methylthio-7-deazainosinic acid) [poly(ms2c7I)] was enzymatically synthesized by polymerization of 2-methylthio-7-deazainosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield. The homopolymer shows much higher thermal stability than its parent polynucleotides poly(7-deazainosinic acid) [poly(c7I)] and poly(I). Its sigmoidal melting curve and pronounced hypochromicity imply a rigid, ordered structure. Poly(ms2c7I), like poly(2-methylthio-inosinic acid) [poly(ms2I)], does not form a complex with poly(C) because of the bulky 2-methylthio substituent. On the other hand, two poly(ms2c7I) strands form very rigid triple strands with poly(A). Different from poly(I) and poly(c7I) the homopolymer poly(ms2c7I) is very stable against cleavage by nuclease S1 and ribonuclease T2 as expected from its rigid secondary structure.
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PMID:Poly(2-methylthio-7-deazainosinic acid)--hydrophobic stabilization of polynucleotide secondary structure by the 2-methylthio group. 688 37

The degradation of individual mRNAs in Escherichia coli has been studied through the use of a multiple mutant carrying the pnp-7 (polynucleotide phosphorylase), rnb-500 (RNase II), and rne-1 (RNase E) alleles. In this triple mutant, discrete mRNA breakdown products are stabilized in vivo at the nonpermissive temperature (Arraiano, C. M., S. D. Yancey, and S. R. Kushner, J. Bacteriol. 170:4625-4633, 1988). In the case of thioredoxin (trxA) mRNA decay, degradation fragments accumulated at early times after a shift to the nonpermissive temperature. Using Northern (RNA) blots, S1 nuclease analysis, and primer extensions, we identified a series of specific endonucleolytic cleavage sites that occur throughout the transcript in both the triple mutant and a wild-type control. The implications of the complex decay patterns observed are discussed.
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PMID:Identification of endonucleolytic cleavage sites involved in decay of Escherichia coli trxA mRNA. 767 84

Messenger RNA decay in Escherichia coli is slowed in pnp-7 (PNPase) rnb-500 (RNase II) rne-1(RNase E) multiple mutants. We have used Northern blots, S1 nuclease protection and primer extension analysis to map 18 endonucleolytic cleavage sites within the pyrF-orfF dicistronic transcript. Although examination of a total of 27 cleavage sites including those determined for the monocistronic trxA transcript revealed a complex pattern, the central four nucleotides within a cluster of 12 residues encompassing the cleavage sites showed a definite A/U preference. Also of interest was the processing of the dicistronic transcript to remove the downstream orfF sequence as a stable but untranslated RNA fragment. The data provide further support for the hypothesis that multiple decay pathways are involved in the decay of a single transcript. In particular, the pyrF-orfF transcript apparently can be degraded either in the 5' to 3' or the 3' to 5' direction. Our results are discussed in light of current models of mRNA decay involving polyadenylation and multiprotein decay complexes.
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PMID:Analysis of the in vivo decay of the Escherichia coli dicistronic pyrF-orfF transcript: evidence for multiple degradation pathways. 915 69

In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Delta26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from Delta26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 3' poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS-, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain Delta26, in which atpB mRNA is unstable because of the lack of a 3' stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PS+ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3'-->5' exonuclease activity, a phenomenon that may occur naturally in the symmetrically transcribed and densely packed chloroplast genome.
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PMID:Antisense transcript and RNA processing alterations suppress instability of polyadenylated mRNA in chlamydomonas chloroplasts. 1548 97