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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 two cleavages made by RNase III in the transcripts of the pnp gene of Escherichia coli, 80 nucleotides upstream of the coding sequence of polynucleotide phosphorylase, were previously demonstrated to trigger the rapid degradation of the pnp messenger. In this paper, we demonstrate that the 5' end of the RNase III processed pnp mRNA is attacked by ribonucleases more efficiently than the rest of the molecule. Several 5' extremities resulting from cleavages occurring in the first 500 nucleotides of the pnp transcript have been identified. Three of them referred to as X, Y and W occur in the wild-type strain at the beginning of the coding sequence of the pnp mRNA. The mRNA appears to be cleaved more efficiently at the X site, proximal to the initiation codon, than at sites Y and W located downstream. In vitro, the maturation at X is catalysed by RNase E but not by RNase III. Accumulation of RNA processed at X in RNase E deficient strains leads us to postulate that X is a high affinity primary site which is slowly cleaved by the residual activity of thermosensitive RNase E at non-permissive temperature and that secondary sites located downstream are processed less efficiently than X. Taken together, our results suggest that in wild-type E. coli the degradation of the RNase III processed mRNA is mediated by RNase E.
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PMID:Nucleolytic inactivation and degradation of the RNase III processed pnp message encoding polynucleotide phosphorylase of Escherichia coli. 751 38

The Escherichia coli rpsO gene gives rise to different mRNA species resulting either from termination of transcription or from processing of primary transcripts by RNase E and RNase III. The main degradation pathway of these transcripts involves a rate-limiting RNase E cleavage downstream of the structural gene which removes the 3' terminal stem-loop structure of the transcription terminator. This structure protects the message from the attack of 3'-5' exonucleases and its removal results in very rapid degradation of the transcript by polynucleotide phosphorylase and RNase II. Polynucleotide phosphorylase is also able to degrade slowly the mRNA harboring the 3' terminal hairpin of the terminator. In contrast, RNase II appears to protect the rpsO mRNA species which retains the 3' hairpin structure. Rapid degradation of the rpsO mRNA is observed after inactivation of RNase II even in a strain deficient for RNase E and polynucleotide phosphorylase. The enzyme(s) involved in this degradation pathway is not known. We detected an unstable elongated rpsO mRNA presumably resulting from the addition of nucleotides at the 3' end of the transcript.
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PMID:Roles of RNase E, RNase II and PNPase in the degradation of the rpsO transcripts of Escherichia coli: stabilizing function of RNase II and evidence for efficient degradation in an ams pnp rnb mutant. 751 47

RNase III is an endonuclease involved in processing both rRNA and certain mRNAs. To help determine whether RNase III (rnc) is required for general mRNA turnover in Escherichia coli, we have created a deletion-insertion mutation (delta rnc-38) in the structural gene. In addition, a series of multiple mutant strains containing deficiencies in RNase II (rnb-500), polynucleotide phosphorylase (pnp-7 or pnp-200), RNase E (rne-1 or rne-3071), and RNase III (delta rnc-38) were constructed. The delta rnc-38 single mutant was viable and led to the accumulation of 30S rRNA precursors, as has been previously observed with the rnc-105 allele (P. Gegenheimer, N. Watson, and D. Apirion, J. Biol. Chem. 252:3064-3073, 1977). In the multiple mutant strains, the presence of the delta rnc-38 allele resulted in the more rapid decay of pulse-labeled RNA but did not suppress conditional lethality, suggesting that the lethality associated with altered mRNA turnover may be due to the stabilization of specific mRNAs. In addition, these results indicate that RNase III is probably not required for general mRNA decay. Of particular interest was the observation that the delta rnc-38 rne-1 double mutant did not accumulate 30S rRNA precursors at 30 degrees C, while the delta rnc-38 rne-3071 double mutant did. Possible explanations of these results are discussed.
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PMID:Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III. 841 98

The rpsO monocistronic messenger, encoding ribosomal protein S15, is destabilized upon polyadenylation occurring at the hairpin structure of the transcription terminator t1. We report that mRNA fragments differing from the monocistronic transcript by their 3' termini are also polyadenylated in the absence of polynucleotide phosphorylase and RNase II. Some of these 3' extremities result from endonucleolytic cleavages by RNase E and RNase III and from exonucleolytic degradation. Most of these mRNA fragments are destabilized upon polyadenylation with the exception of the RNA species generated by RNase III. RNase E appears to reduce the amount of poly(A) added at the transcription terminator t1.
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PMID:The rpsO mRNA of Escherichia coli is polyadenylated at multiple sites resulting from endonucleolytic processing and exonucleolytic degradation. 867 Aug 15

ColE1 DNA replication is initiated by RNA II and inhibited by RNA I. Control of the replication occurs through the interaction between RNA I and RNA II. Therefore, RNases involved in the metabolism of RNA I and RNA II are expected to play a key role in the control of the ColE1 plasmid replication. RNase H, RNase E, RNase III, RNase P, and polynucleotide phosphorylase carry out the many specific reactions of the RNA metabolism.
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PMID:RNases in ColE1 DNA metabolism. 890 10

The degradation process of the rpsO mRNA is one of the best characterised in E coli. Two independent degradation pathways have been identified. The first one is initiated by an RNase E endonucleolytic cleavage which allows access to the transcript by polynucleotide phosphorylase and RNase II. Cleavage by RNase E gives rise to an rpsO message lacking the stabilising hairpin of the primary transcript; this truncated mRNA is then degraded exonucleolytically from its 3' terminus. This pathway might be coupled to the translation of the message. The second pathway allows degradation of polyadenylated rpsO mRNA independently of RNase II, PNPase and RNase E. The ribonucleases responsible for degradation of poly(A) mRNAs under these conditions are not known. Poly(A) tails have been proposed to facilitate the degradation of structured RNA by polynucleotide phosphorylase. In contrast, we believe that removal of poly(A) by RNase II stabilises the rpsO mRNA harbouring a 3' hairpin. In addition to these two pathways, we have identified endonucleolytic cleavages which occur only in strains deficient for both RNase E and RNase III suggesting that these two endonucleases protect the 5' leader of the mRNA from the attack of unidentified ribonuclease(s). Looping of the rpsO mRNA might explain how RNase E bound at the 5' end can cleave at a site located just upstream the hairpin of the transcription terminator.
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PMID:Multiple degradation pathways of the rpsO mRNA of Escherichia coli. RNase E interacts with the 5' and 3' extremities of the primary transcript. 891 31

Metabolic instability is a hallmark property of mRNAs in most if not all organisms and plays an essential role in facilitating rapid responses to regulatory cues. This article provides a critical examination of recent progress in the enzymology of mRNA decay in Escherichia coli, focusing on six major enzymes: RNase III, RNase E, polynucleotide phosphorylase, RNase II, poly(A) polymerase(s), and RNA helicase(s). The first major advance in our thinking about mechanisms of RNA decay has been catalyzed by the possibility that mRNA decay is orchestrated by a multicomponent mRNA-protein complex (the "degradosome"). The ramifications of this discovery are discussed and developed into mRNA decay models that integrate the properties of the ribonucleases and their associated proteins, the role of RNA structure in determining the susceptibility of an RNA to decay, and some of the known kinetic features of mRNA decay. These models propose that mRNA decay is a vectorial process initiated primarily at or near the 5' terminus of susceptible mRNAs and propagated by successive endonucleolytic cleavages catalyzed by RNase E in the degradosome. It seems likely that the degradosome can be tethered to its substrate, either physically or kinetically through a preference for monphosphorylated RNAs, accounting for the usual "all or none" nature of mRNA decay. A second recent advance in our thinking about mRNA decay is the rediscovery of polyadenylated mRNA in bacteria. Models are provided to account for the role of polyadenylation in facilitating the 3' exonucleolytic degradation of structured RNAs. Finally, we have reviewed the documented properties of several well-studied paradigms for mRNA decay in E. coli. We interpret the published data in light of our models and the properties of the degradosome. It seems likely that the study of mRNA decay is about to enter a phase in which research will focus on the structural basis for recognition of cleavage sites, on catalytic mechanisms, and on regulation of mRNA decay.
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PMID:Degradation of mRNA in Escherichia coli: an old problem with some new twists. 993 52

A 320-nucleotide RNA with several characteristic features was expressed in Bacillus subtilis to study RNA processing. The RNA consisted of a 5'-proximal sequence from bacteriophage SP82 containing strong secondary structure, a Bs-RNase III cleavage site, and the 3'-proximal end of the ermC transcriptional unit. Comparison of RNA processing in a wild-type strain and a strain in which the pnpA gene, coding for polynucleotide phosphorylase (PNPase), was deleted, as well as in vitro assays of phosphate-dependent degradation, showed that PNPase activity could be stalled in vivo and in vitro. Analysis of mutations in the SP82 moiety mapped the block to PNPase processivity to a particular stem-loop structure. This structure did not provide a block to processivity in the pnpA strain, suggesting that it was specific for PNPase. An abundant RNA with a 3' end located in the ermC coding sequence was detected in the pnpA strain but not in the wild type, indicating that this block is specific for a different 3'-to-5' exonuclease. The finding of impediments to 3'-to-5' degradation, with specificities for different exonucleases, suggests the existence of discrete intermediates in the mRNA decay pathway.
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PMID:Protection against 3'-to-5' RNA decay in Bacillus subtilis. 1057 37

RNAI is a short RNA, 108 nt in length, which regulates the replication of the plasmid ColE1. RNAI turns over rapidly, enabling plasmid replication rate to respond quickly to changes in plasmid copy number. Because RNAI is produced in abundance, is easily extracted and turns over quickly, it has been used as a model for mRNA in studying RNA decay pathways. The enzymes polynucleotide phosphorylase, poly(A) polymerase and RNase E have been demonstrated to have roles in both messenger and RNAI decay; it is reported here that these enzymes can work independently of one another to facilitate RNAI decay. The roles in RNAI decay of two further enzymes which facilitate mRNA decay, the exonuclease RNase II and the endonuclease RNase III, are also examined. RNase II does not appear to accelerate RNAI decay but it is found that, in the absence of RNase III, polyadenylated RNAI, unprocessed by RNase E, accumulates. It is also shown that RNase III can cut RNAI near nt 82 or 98 in vitro. An RNAI fragment corresponding to the longer of these can be found in extracts of an mc+ pcnB strain (which produces RNase III) but not of an rnc pcnB strain, suggesting that RNAI may be a substrate for RNase III in vivo. A possible pathway for the early steps in RNAI decay which incorporates this information is suggested.
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PMID:Absence of RNASE III alters the pathway by which RNAI, the antisense inhibitor of ColE1 replication, decays. 1058 16

To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half-life of total pulse-labelled RNA along with decreased half-lives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. Long-term overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3'-terminus of 23S rRNA, a site that was not tailed in wild-type cells. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated an almost 20-fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript-dependent manner.
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PMID:Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism. 1059 33


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