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

RNase II of Escherichia coli (EC 3.1.4.23) has been purified to apparent homogeneity. The K+-activated diesterase activity against poly(U), which defines RNase II, cochromatographs with activity against T4 mRNA or pulse-labeled E. coli RNA successively on DEAE-cellulose, hydroxyapatite or phosphocellulose, and Sephadex G-150 columns. Activities with both substrates are selectively reduced to less than 2% of the wild type level in a newly isolated mutant strain, S296, or after thermal inactivation in a mutant strain with temperature-sensitive RNase II. RNase II releases 5'-XMP without a lag as its only detectable alcohol-soluble produce from all substrates and has an apparent molecular weight of 80,000 to 90,000 in both nondissociating and sodium dodecyl sulfate-polyacrylamide gels. The pure enzyme shows the standard K+ activation against poly(A), poly(U), or poly(C), but only a slight preference for K+ over Na+ ions with T4 mRNA or pulse labeled E. coli RNA as substrate. Uniformly labeled E. coli rRNA or tRNA is degraded little if at all.
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PMID:Purification and some novel properties of Escherichia coli RNase II. 33 25

Ribonucleases O and Q, the two putative nucleolytic activities which we detected previously in the crude extract from a thermosensitive ribonuclease P mutant (TS241) of Escherichia coli and which were shown to function in the processing of tRNA precursors in vitro, were partially purified from the 1000000 x g supernatant fraction of E. coli Q13. In the course of purification of these enzymes, the total RNAs synthesized in the thermosensitive mutant at the restrictive temperature were used as the substrates and the activities were identified from disappearance or alteration of specific tRNA precursor molecules in polyacrylamide gel electrophoresis. The purified ribonuclease O preparation cleaved specifically the multimeric tRNA precursors at the spacer regions. The purified ribonuclease Q preparation removed, in accordance with the definition of this enzyme, extra nucleotides from the 3'-terminal ends of monomeric tRNA precursors. Some properties of these two nucleases were investigated. In addition to these nucleases, another exonuclease (tentatively designated ribonuclease Y) and ribonuclease P, a well-characterized endonuclease, were also purified. The sequential mode of the processing of tRNA precursors, originally observed in the cleavage reactions with the crude extracts in vitro, was supported by studies with the purified enzyme preparations.
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PMID:Specific ribonucleases involved in processing of tRNA precursors of Escherichia coli. Partial purification and some properties. 35 May 82

The synthetic tRNA precursors, tRNA-C-114C]U and tRNA-C-C-A-[14C]C-C, as well as poly (a) and diesterase-treated tRNA, have been used to identify and purify potential 3'processing nucleases. Four activities have been separated by this analysis; and three of them have been characterized. Two of the enzymes, which are well-separated on hydroxylapatite columns, act on poly(A), require K+ and Mg2+ for activity, and have molecular weights of about 90,000. These activities have properties previously ascribed to RNase II. The third enzyme does not act on poly(A), requires Mg2+ for activity, and has a molecular weight of about 60,000. It is identical to RNase D, previously characterized as an exonuclease acting on tRNAs with altered structure. Each of the enzymes can remove nucleotides from the tRNA precursor containing extra nucleotides beyond the 3'terminus, whereas they are relatively inactive with intact tRNA or tRNA-C-U. The greatest specificity was displayed by RNase D. The possibility that RNase D is a 3'processing nuclease is discussed.
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PMID:Purification of potential 3' processing nucleases using synthetic tRNA precursors. 36 19

Our results indicate that RNase P has a very general role in the processing of tRNA precursors in E. coli, being responsible for the cleavage of virtually all precursor molecules at a site corresponding to the 5' end of the mature tRNA, and that at least two other RNases play specific roles in precursor processing. One of these, which may be RNase II, is responsible for removing extra nucleotides from the 3' end of tRNA precursors. The other, which we call RNase P2, is an endonuclease that cleaves precursors in spacer regions between different tRNA sequences; this enzyme is involved in the processing of large multimeric precursors.
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PMID:Processing of E. coli tRNA precursors. 110

RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro. To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains. Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase. Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability. The rph mutation also had dramatic effects on tRNA metabolism. Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases. Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence. These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors.
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PMID:RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells. 164 89

Escherichia coli RNase PH is a phosphate-dependent exoribonuclease that has been implicated in the 3' processing of tRNA precursors. It degrades RNA chains in a phosphorolytic manner releasing nucleoside diphosphates as products. Here we show that RNase PH also catalyzes a synthetic reaction, the addition of nucleotides to the 3' termini of RNA molecules. The synthetic activity co-purifies with RNase PH throughout an extensive enrichment indicating that it is due to the same enzyme. The synthetic activity can incorporate all nucleoside diphosphates, but not triphosphates, and is strongly inhibited by Pi, but not PPi. Various RNA molecules stimulate nucleotide incorporation, and with tRNA the 3' end of the molecule serves a primer function. RNA chains as long as 40 residues can be synthesized in this system. As with polynucleotide phosphorylase, the synthetic activity of RNase PH apparently represents the reversal of the degradative reaction.
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PMID:RNase PH catalyzes a synthetic reaction, the addition of nucleotides to the 3' end of RNA. 170 83

RNase T, a nuclease thought to be involved in end-turnover of tRNA, has been purified about 4,000-fold from extracts of Escherichia coli. At this stage of purification, the enzyme was judged to be at least 95% pure based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight of RNase T determined from gel filtration and sedimentation analyses is about 50,000, whereas the monomer molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25,000, suggesting that the protein is an alpha 2 dimer. Purified RNase T is extremely sensitive to inactivation by oxidation, sulfhydryl group reagents, and temperature. The ribonuclease activity against tRNA-C-C-[14C]A is optimal at pH 8-9 in the presence of 2-5 mM MgCl2 and ionic strengths of less than 50mM. Although RNase T is highly specific for intact tRNA-C-C-A as a substrate and can hydrolyze all species in a mixed population of tRNA, it is inhibited by other RNAs, such as poly(A), rRNA, 5 S RNA, and tRNA-C-C. RNase T is an exoribonuclease which initiates attack at a free 3' terminus of tRNA and releases AMP; aminoacyl-tRNA is not a substrate. The role of RNase T in the end-turnover of tRNA and its possible involvement in other aspects of RNA metabolism are discussed.
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PMID:Purification and characterization of Escherichia coli RNase T. 388 94

Escherichia coli RNase D and RNase II have been purified to homogeneity and compared for their ability to remove extra nucleotides following the -C-C-A sequence in tRNA precursors. RNase D and RNase II are single-chain proteins with molecular weights of 38,000 and 78,000, respectively. Both enzymes require a divalent cation for activity on tRNA precursors, but, in addition, RNase II is stimulated by monovalent cations. RNase D specifically removes mononucleotide residues from a mixture of tRNA precursors to generate amino acid acceptor activity for essentially all amino acids. Although RNase II can also remove precursor-specific residues, no amino acid acceptor activity is recovered. Similarly, RNase D action on the E. coli tRNATyr precursor is limited, whereas RNase II causes extensive degradation. In contrast to the processive mode of hydrolysis by RNase II, RNase D removes nucleotides randomly and slows down greatly at the -C-C-A sequence, thereby allowing the tRNA to be aminoacylated and protected from further degradation. These results suggest that RNase D is the 3'-processing nuclease in vivo and that RNase II is a nonspecific degradative enzyme. The importance of RNA conformation for correct processing is also discussed.
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PMID:Apparent involvement of ribonuclease D in the 3' processing of tRNA precursors. 615 5

A new ribonuclease, RNase BN, has been identified and partially purified from a strain of Escherichia coli lacking RNase II and RNase D by using the artificial tRNA precursor tRNA-C-[14C]U as substrate. This enzyme is present in E. coli B but absent from the tRNA processing mutant strain BN which is unable to process extraneous 3' residues on certain phage T4-specified tRNA precursors. The properties of RNase BN clearly distinguish this enzyme from other known E. coli exoribonucleases. It is optimally active at pH 6.5 with 0.2 mM divalent cation and 0.2 M monovalent cation. It is most active against tRNA substrates containing nucleotide substitutions within the -C-C-A sequence and relatively inactive against other types of RNAs. This substrate specificity in vitro is consistent with a processing function in vivo. However, in contrast to the other processing enzymes whose function has been confirmed by mutation, RNase BN is an exoribonuclease. The presence of multiple RNases in E. coli and a strategy for their identification and separation are discussed.
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PMID:Ribonuclease BN: identification and partial characterization of a new tRNA processing enzyme. 634 80

Examination of double mutants lacking one of the exoribonucleases, RNase II, RNase D, RNase BN, or RNase R, and also devoid of tRNA nucleotidyltransferase has suggested that none of these RNases participates in the end-turnover of tRNA. This prompted a search for and identification of a new exoribonuclease, termed RNase T. RNase T could be detected in mutant Escherichia coli strains lacking as many as three of the known exoribonucleases, and it could be separated from each of the four previously described RNases. RNase T is optimally active at pH 8-9 and requires a divalent cation for activity. The enzyme is sensitive to ionic strengths greater than 50 mM and is rapidly inactivated by heating at 45 degrees C. Its preferred substrate is tRNA-C-C-[14C]A, with much less activity shown against tRNA-C-C. RNase T is an exoribonuclease that initiates attack at the 3' hydroxyl terminus of tRNA and releases AMP in a random mode of hydrolysis. The possible involvement of RNase T in end-turnover of tRNA and in RNA metabolism in general are discussed.
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PMID:Ribonuclease T: new exoribonuclease possibly involved in end-turnover of tRNA. 637 42


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