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:3.1.13.1 (exoribonuclease)
732 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed.
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PMID:Proteins associated with RNase E in a multicomponent ribonucleolytic complex. 863 81

The 3'-exonucleolytic decay of the mRNA for ribosomal protein S20 has been reconstituted in vitro using purified RNase II and crude extracts enriched for polynucleotide phosphorylase (PNPase) activity. We show that RNase II can catalyze the degradation of the 5' two-thirds of the S20 mRNA and that prior oligoadenylation of the 3' termini of truncated S20 mRNA substrates can significantly stimulate the initiation of degradation by RNase II. The intact S20 mRNA is, however, insensitive to attack by RNase II and polyadenylation of its 3'-end cannot overcome the natural resistance of the S20 mRNA to RNase II. Complete degradation of either the entire S20 mRNA without prior endonucleolytic cleavage or the 3'-terminal 147-residue fragment is dependent on both oligoadenylation and PNPase activity. Moreover, this process can take place in the absence of RNase E activity. Our data point to the importance of oligoadenylation in facilitating 3'-exonucleolytic activity and indicate that there are alternative degradative pathways. The implications for mRNA decay are discussed.
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PMID:Differential sensitivities of portions of the mRNA for ribosomal protein S20 to 3'-exonucleases dependent on oligoadenylation and RNA secondary structure. 866 15

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

The monocistronic transcript of rpsO undergoes an endonucleolytic cleavage downstream of the coding sequence, which removes the hairpin of the transcription terminator and initiates the rapid degradation of the message. We demonstrate here that the two rne-dependent cleavages, on both sides of the transcription terminator, are catalysed by RNase E in vitro and that the RNase E-processed rpsO message is rapidly degraded by polynucleotide phosphorylase, while RNase II produces stable decay intermediates. Moreover, we show that RNase E cuts in vitro the coding sequence of the rpsO mRNA at several sites which are not detected in vivo.
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PMID:Polynucleotide phosphorylase is required for the rapid degradation of the RNase E-processed rpsO mRNA of Escherichia coli devoid of its 3' hairpin. 883 Feb 80

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

The S1 domain, originally identified in ribosomal protein S1, is found in a large number of RNA-associated proteins. The structure of the S1 RNA-binding domain from the E. coli polynucleotide phosphorylase has been determined using NMR methods and consists of a five-stranded antiparallel beta barrel. Conserved residues on one face of the barrel and adjacent loops form the putative RNA-binding site. The structure of the S1 domain is very similar to that of cold shock protein, suggesting that they are both derived from an ancient nucleic acid-binding protein. Enhanced sequence searches reveal hitherto unidentified S1 domains in RNase E, RNase II, NusA, EMB-5, and other proteins.
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PMID:The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold. 900 64

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

The Escherichia coli degradosome is a multienzyme complex with four major protein components: the endoribonuclease RNase E, the exoribonuclease PNPase, the RNA helicase RhlB and enolase. The first three of these proteins are known to have important functions in mRNA processing and degradation. In this work, we identify an additional component of the degradosome, polyphosphate kinase (PPK), which catalyses the reversible polymerization of the gamma-phosphate of ATP into polyphosphate (poly(P)). An E. coli strain deleted for the ppk gene showed increased stability of the ompA mRNA. Purified His-tagged PPK was shown to bind RNA, and RNA binding was prevented by hydrolysable ATP. Chemical modification of RNA by PPK, for example the addition or removal of 3' or 5' terminal phosphates, could not be detected. However, polyphosphate was found to inhibit RNA degradation by the degradosome in vitro. This inhibition was overcome by the addition of ADP, required for the degradation of polyphosphate and for the regeneration of ATP by PPK in the degradosome. Thus, PPK in the degradosome appears to maintain an appropriate microenvironment, removing inhibitory polyphosphate and NDPs and regenerating ATP.
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PMID:Polyphosphate kinase is a component of the Escherichia coli RNA degradosome. 938 62

Polyribonucleotide phosphorylase (PNPase) is one of the critical components of the E. coli RNA degradosome, which consists of both PNPase and endoribonuclease RNase E. The function of this complex is to control the rate of mRNA degradation. The PNPase possesses two enzymatic activities, namely 3'-5' processive exoribonuclease activity and 5'-3' RNA polymerase activity. In the present study, we used conventional chromatography to purify an E. coli protein that binds to a specific double-stranded DNA sequence. Microsequencing of the purified protein showed that this DNA-binding protein was PNPase. Our data further demonstrate that PNPase binds to DNA in a sequence-specific manner. These data suggest that PNPase may have previously unappreciated DNA-related functions in addition to its known role in mRNA degradation.
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PMID:Polyribonucleotide phosphorylase is a double-stranded DNA-binding protein. 950 33


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