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Dawid G?ów Dariusz Pianka Agata A. Sulej ?ukasz P. Koz?owski Justyna Czarnecka Grzegorz Chojnowski Krzysztof J. Skowronek Janusz M. Bujnicki 《Nucleic acids research》2015,43(5):2864-2873
Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner. Here, we present evidence for a sequence-dependent cleavage of long dsRNA by RNase Mini-III from Bacillus subtilis (BsMiniIII). Analysis of the sites cleaved by this enzyme in limited digest of bacteriophage Φ6 dsRNA led to the identification of a consensus target sequence. We defined nucleotide residues within the preferred cleavage site that affected the efficiency of the cleavage and were essential for the discrimination of cleavable versus non-cleavable dsRNA sequences. We have also determined that the loop α5b-α6, a distinctive structural element in Mini-III RNases, is crucial for the specific cleavage, but not for dsRNA binding. Our results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be used for targeted cleavage of dsRNA. 相似文献
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RNase E, an RNA processing enzyme from Escherichia coli. 总被引:18,自引:0,他引:18
An activity, RNase E, was purified about 100-fold from Escherichia coli cells, it can process p5 rRNA from a 9 S RNA molecule which accumulates in a mutant of E. coli defective in the maturation of 5 S rRNA. The enzyme requires Na+, K+, or NH4+, and Mg2+ or Mn2+. The molecular weight of the enzyme is about 70,000 and its pH optimum is 7.6 to 8.0. Its temperature optimum is around 30 degrees C, and it can be irreversibly inactivated at 50 degrees C. It has a very high degree of specificity but the reaction can be inhibited by nonspecific RNAs. We interpret its mode of action in producing p5 RNA as being accomplished in two steps, 9 S RNA is first processed to 7 S and 4 S, and subsequently 7 S is further processed to p5. 相似文献
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Multiprotein complexes that carry out RNA degradation and processing functions are found in cells from all domains of life. In Escherichia coli, the RNA degradosome, a four-protein complex, is required for normal RNA degradation and processing. In addition to the degradosome complex, the cell contains other ribonucleases that also play important roles in RNA processing and/or degradation. Whether the other ribonucleases are associated with the degradosome or function independently is not known. In the present work, IP (immunoprecipitation) studies from cell extracts showed that the major hydrolytic exoribonuclease RNase II is associated with the known degradosome components RNaseE (endoribonuclease E), RhlB (RNA helicase B), PNPase (polynucleotide phosphorylase) and Eno (enolase). Further evidence for the RNase II-degradosome association came from the binding of RNase II to purified RNaseE in far western affinity blot experiments. Formation of the RNase II–degradosome complex required the degradosomal proteins RhlB and PNPase as well as a C-terminal domain of RNaseE that contains binding sites for the other degradosomal proteins. This shows that the RNase II is a component of the RNA degradosome complex, a previously unrecognized association that is likely to play a role in coupling and coordinating the multiple elements of the RNA degradation pathways. 相似文献
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Two models have been proposed for triggering release of the lagging strand polymerase at the replication fork, enabling cycling to the primer for the next Okazaki fragment—either collision with the 5′-end of the preceding fragment (collision model) or synthesis of a new primer by primase (signaling model). Specific perturbation of lagging strand elongation on minicircles with a highly asymmetric G:C distribution with ddGTP or dGDPNP yielded results that confirmed the signaling model and ruled out the collision model. We demonstrated that the presence of a primer, not primase per se, provides the signal that triggers cycling. Lagging strand synthesis proceeds much faster than leading strand synthesis, explaining why gaps between Okazaki fragments are not found under physiological conditions. 相似文献
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RNase E is an essential endoribonuclease involved in RNA processing and mRNA degradation. The N-terminal half of the protein encompasses the catalytic domain; the C-terminal half is the scaffold for the assembly of the multienzyme RNA degradosome. Here we identify and characterize 'segment-A', an element in the beginning of the non-catalytic region of RNase E that is required for membrane binding. We demonstrate in vitro that an oligopeptide corresponding to segment-A has the propensity to form an amphipathic alpha-helix and that it avidly binds to protein-free phospholipid vesicles. We demonstrate in vitro and in vivo that disruption of segment-A in full-length RNase E abolishes membrane binding. Taken together, our results show that segment-A is necessary and sufficient for RNase E binding to membranes. Strains in which segment-A has been disrupted grow slowly. Since in vitro experiments show that phospholipid binding does not affect the ribonuclease activity of RNase E, the slow-growth phenotype might arise from a defect involving processes such as accessibility to substrates or interactions with other membrane-bound machinery. This is the first report demonstrating that RNase E is a membrane-binding protein and that its localization to the inner cytoplasmic membrane is important for normal cell growth. 相似文献
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Eric J. Montemayor Adam Katolik Nathaniel E. Clark Alexander B. Taylor Jonathan P. Schuermann D. Joshua Combs Richard Johnsson Stephen P. Holloway Scott W. Stevens Masad J. Damha P. John Hart 《Nucleic acids research》2014,42(16):10845-10855
The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2′,5′-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2′,5′-phosphodiester recognition and explain why the enzyme lacks activity toward 3′,5′-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease. 相似文献
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The rne gene is the structural gene for the processing endoribonuclease RNase E of Escherichia coli.
Summary Using T7 RNA polymerase and specific constructs derived from 5S rRNA and RNA I genes, we generated substrates for the RNA processing enzyme RNase E. Using these substrates we have shown that a 3.2 kb DNA fragment that complements the rne-3071 mutation can express RNase E activity. We also found that T7 RNA polymerase terminates within the 5S rRNA gene. 相似文献
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We recently showed that RNase III can process a small stable RNA, precursor 10Sa RNA, that accumulates in an rne (RNase E) strain at non-permissive temperatures. Precursor 10Sa (p10Sa) RNA is processed to 10Sa RNA in two steps, the first step is catalyzed by RNase III in the presence of Mn2+ but not Mg2+. It was shown that RNase III cosediments with membrane preparation from wild type as well as RNase III overexpressing cells. However, the possibility of membrane preparation contamination with ribosomes could not be ruled out. Here we show that RNase III, E and P are not associated with ribosomes. E. coli cells were opened either by alumina grinding or by sonication and fractionated into cytosolic and pellet fractions. The characterization of membrane preparations was done by assaying NADH oxidase, a bona fide membrane enzyme. Ribosomes prepared by alumina grinding were found to be contaminated with small fragments of membrane which contained RNase III activity. RNase III and NADH oxidase activities were present in the ribosomal preparations which could be solubilized by reagents that dissolve the inner membrane. Isopycnic sucrose gradient centrifugation of the membrane and ribosomal preparations also confirmed that RNase III fractionated with the inner membrane. Similarly RNase P activity was found in the corresponding fractions when isopycnic centrifugation of membrane and ribosome preparations was carried out. RNase E activity was also found to be present mostly in the post-ribosomal supernatant. These findings show that RNase III, E and P are not ribosomal enzymes. 相似文献
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Abstract The speA gene encoding streptococcal erythrogenic toxin A (SPE A) from Streptococcus pyogenes bacteriophage T12 was overexpressed in Escherichia coli under the control of the T7 promoter. Since most of the expressed protein was found in the periplasmic space, an osmotic shock extraction with 0.5 M sucrose resulted in a highly enriched preparation of SPE A. An additional two-step purification employing high pressure liquid chromatography resulted in a purified SPE A protein. 相似文献
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Callaghan AJ Aurikko JP Ilag LL Günter Grossmann J Chandran V Kühnel K Poljak L Carpousis AJ Robinson CV Symmons MF Luisi BF 《Journal of molecular biology》2004,340(5):965-979
The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10-40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein-RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed. 相似文献
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Escherichia coli cells normally require RNase E activity to propagate and form colonies. Using random Tn10 insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) to E. coli cells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of the rne mutant phenotype by inactivation of deaD was partial, as rne deaD doubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration by deaD inactivation requires normal expression of the endogenous rng gene in doubly mutant rne deaD cells. Second-site suppression by deaD mutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality. 相似文献
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Transfer RNA precursors are accumulated in Escherichia coli in the absence of RNase E 总被引:8,自引:0,他引:8
A temperature-sensitive Escherichia coli mutant, which contains a heat-labile RNase E, fails to produce 5-S rRNA at a non-permissive temperature. It accumulates a number of RNA molecules in the 4-12-S range. One of these molecules, a 9-S RNA, is a precursor to 5-S rRNA [Ghora, B. K. and Apirion, D. (1978) Cell, 15, 1055-1056]. These molecules were purified and processed in a cell-free system. Some of these RNA molecules, after processing, give rise to products the size of transfer RNA, but not to 5-S-rRNA. Further characterization of the processed products of one such precursor molecule shows that it contains tRNA1Leu and tRNA1His. RNase E is necessary but not sufficient for the processing of this molecule to mature tRNAs in vitro. The accumulation of such tRNA precursors in an RNase E mutant cell and the obligatory participation of RNase E in its processing indicate that RNase E functions in the maturation of transfer RNAs as well as of 5-S rRNA. 相似文献