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At least 865 records · Page 48Linked to original sources
PubMed1991

Sequence changes in both flanking sequences of a pre-tRNA influence the cleavage specificity of RNase P.

The cleavage specificities of the RNase P holoenzymes from Escherichia coli and the yeast Schizosaccharomyces pombe and of the catalytic M1 RNA from E. coli were analyzed in 5'-processing experiments using a yeast serine pre-tRNA with mutations in both flanking sequences. The template DNAs were obtained by enzymatic reactions in vitro and transcribed with phage SP6 or T7 RNA polymerase. The various mutations did not alter the cleavage specificity of the yeast RNase P holoenzyme; cleavage always occurred predominantly at position G + 1, generating the typical seven base-pair acceptor stem. In contrast, the specificity of the prokaryotic RNase P activities, i.e. the catalytic M1 RNA and the RNase P holoenzyme from E. coli, was influenced by some of the mutated pre-tRNA substrates, which resulted in an unusual cleavage pattern, generating extended acceptor stems. The bases G - 1 and C + 73, forming the eighth base pair in these extended acceptor stems, were an important motif in promoting the unusual cleavage pattern. It was found only in some natural pre-tRNAs, including tRNA(SeCys) from E. coli, and tRNAs(His) from bacteria and chloroplasts. Also, the corresponding mature tRNAs in vivo contain an eight base pair acceptor stem. The presence of the CCA sequence at the 3' end of the tRNA moiety is known to enhance the cleavage efficiency with the catalytic M1 RNA. Surprisingly, the presence or absence of this sequence in two of our substrate mutants drastically altered the cleavage specificity of M1 RNA and of the E. coli holoenzyme, respectively. Possible reasons for the different cleavage specificities of the enzymes, the influence of sequence alterations and the importance of stacking forces in the acceptor stems are discussed.

Base Sequence↗
PubMed1992

Structural requirements for viroid processing by RNase T1.

Viroids are replicated via a rolling circle-like mechanism in which (+) strand oligomeric intermediates have to be cleaved enzymatically to unit-length molecules followed by ligation to mature circles. A transcript of potato spindle tuber viroid, which is still infectious, consists of a monomeric molecule with only 22 additional nucleotides, thus doubling part of the central conserved region of viroids. It was shown that this transcript can be cleaved and ligated in vitro to circles by RNase T1. To elucidate the site and mechanism of processing, 16 different site-specific mutants of this longer-than-unit-length transcript were constructed and analyzed by in vitro processing with RNase T1, infectivity studies, temperature-gradient gel electrophoresis, and structure calculations. The wild-type sequence and several mutated transcripts are able to adopt a particular secondary structure which is the prerequisite for enzymatic cleavage and ligation by RNase T1. This "processing structure" exposes both potential cleavage sites in the nearest spatial neighborhood, thus favoring the subsequent ligation to circles. Those mutated sequences for which the formation of the processing structure is impossible or thermodynamically highly unfavored are not processed. The results demonstrate that the particular structural features of viroids enable them to be cleaved and ligated by one and the same enzyme, RNase T1. The in vitro mechanism may serve as a mechanistic model for cellular processing of viroids.

Base Sequence↗
PubMed1989

(2'-5')An-dependent RNase: enzyme levels are decreased in barrier-reared control and IFN-beta or IFN-gamma treated Balb/c mice.

(2'-5')An-dependent RNase functions as a translational regulatory protein which mediates interferon action. Levels of this enzyme are decreased in barrier-reared Balb/c (+/+), Balb/c (+/nu), and Balb/c (nu/nu) mice when compared to conventionally reared Balb/c (+/+) mice. This suggests that high levels of (2'-5')An-dependent RNase in conventionally reared mice are maintained by continuous exposure to microbial flora which may induce interferons. Interferon treatment of barrier-reared mice does not, however, result in an increase in (2'-5')An-dependent RNase levels. This suggests that responsiveness to interferons is decreased in barrier-reared mice. The high levels of (2'-5')An-dependent RNase which are maintained in normal mice under physiological conditions may be important for rapid and effective defense against viral pathogens.

Animals↗
PubMed1993

Estimates by computer simulation of genetic distances from comparisons of RNAse A mismatch cleavage patterns.

The RNAse A mismatch cleavage method was used to analyze genomic variability in RNA and DNA systems. However, there is no method which relates the digestion patterns observed to the extent of genetic variation. Here we report computer simulations which provide a simple estimator of genetic distances from the comparison of RNAse A digestion patterns. The results show that the number of non-shared fragments is proportional to the number of mutations between each pair of sequences compared. This prediction is supported by the comparison of the RNAse A mismatch patterns and the nucleotide sequences of a set of influenza A (H3N2) hemagglutinin genes. The procedure allows a quantitative and reliable use of the RNAse A mismatch cleavage method.

Computer Simulation↗
PubMed1994

Characterization of an RNase H deficient mutant of human immunodeficiency virus-1 reverse transcriptase having an aspartate to asparagine change at position 498.

It has been proposed that Asp-443, Glu-478, and Asp-498 are important in RNase H mediated catalysis by human immunodeficiency virus-1 reverse transcriptase (Davies J.F., Hostomska, Z. Hostomsky, Z., Jordan, S.R. and Matthews, D.A. (1991) Science 251, 88-95; Mizrahi, V., Usdin, M.T., Harington, A. and Dudding, L.R. (1990) Nucleic Acids Res. 18, 5359-5363). Single point mutations at either position 443 (Mizrahi, V., Usdin, M.T., Harington, A. and Dudding, L.R. (1990) Nucleic Acids Res. 18, 5359-5363) or 478 (Schatz, O., Cromme, F.V., Grüninger-Leitch, F. and Le Grice, S.F.J. (1989) FEBS Lett. 257, 311-314) severely inhibit RNase H activity but have only small effects on polymerase activity. We show here that a single mutation at position 498 of Asp to Asn (mutant D498N) results in a stable enzyme with a 20-fold reduction in the ratio of RNase H to polymerase activity. The mutant and wild type enzymes were equally processive, paused in the same locations, and extended primers at the same rate during DNA synthesis on a heteropolymeric RNA template. The rate of elongation on the homopolymeric template poly(rA) was also the same. The results indicate that the mutation does not affect normally measured catalytic parameters of the polymerase function of the enzyme. D498N catalyzed strand transfer synthesis on homopolymeric, but not heteropolymeric templates, indicating that RNase H activity is not required for the former activity, but is for the latter.

Asparagine↗
PubMed1990

RNase PH catalyzes a synthetic reaction, the addition of nucleotides to the 3' end of RNA.

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.

Exoribonucleases↗
PubMed1993

Phosphorothioates in pre-tRNAs can change the specificities of RNAses P or reduce the cleavage efficiencies.

Several phosphorothioate-modified E coli and yeast pre-tRNAs were synthesized. If this modification included the phosphodiester at the RNase P cleavage site, two different effects were observed. With some pre-tRNAs the RNase P cleavage efficiency was severely reduced, whereas with other pre-tRNAs a new reaction type for RNase P was observed. Unlike the previously studied base or ribose modifications, phosphorothioates resulted in aberrant cleavages at unmodified phosphodiesters. These new sites could be located in the 5'-flank or in the acceptor stem of the tRNA domain. Modified mutants of E coli pre-tRNA(Tyr) with different base pairs at the RNase P cleavage site were cleaved with reduced efficiencies, but no aberrant products were observed.

Base Composition↗
PubMed1993

Processing of the Pseudomonas arcDABC mRNA requires functional RNase E in Escherichia coli.

In Pseudomonas aeruginosa, arginine catabolism via the arginine deiminase pathway depends on the anaerobically inducible arcDABC operon, whose expression is further modulated by mRNA processing. Fusion of the cloned arc operon to an external tac promoter did not alter the processing pattern in P. aeruginosa and allowed heterologous expression in Escherichia coli. Processing within a specific region of the arcD mRNA was similar in P. aeruginosa and in E. coli. In E. coli, a conditional temperature-sensitive (ts) mutation in the gene specifying RNase E prevented cleavage of the arc mRNA at the non-permissive temperature, whereas mutations in the genes encoding RNase III or RNase P had no effect. We therefore speculate that in P. aeruginosa, an RNase E-like enzyme exists which is involved in the specific processing of the arc mRNA.

Amino Acid Transport Systems↗
PubMed1991

Seminal RNase: a unique member of the ribonuclease superfamily.

The RNase found in bull semen, although a member of the mammalian superfamily of ribonucleases, possesses some unusual properties. Besides its unique structure and enzymic properties, it displays antispermatogenic, antitumor and immunosuppressive activities. Seminal RNase belongs to an interesting group of RNases, the RISBASES (RIbonucleases with Special, i.e. non catalytic, Biological Actions) other members of which include angiogenin, selectively neurotoxic RNases, a lectin and the self-incompatibility factors from a flowering plant.

Animals↗
PubMed2005

Secondary structure probing of the human RNase MRP RNA reveals the potential for MRP RNA subsets.

RNase MRP is a ribonucleoprotein endoribonuclease involved in eukaryotic pre-rRNA processing. The enzyme possesses an RNA subunit, structurally related to that of RNase P RNA, that is thought to be catalytic. RNase MRP RNA sequences from Saccharomycetaceae species are structurally well defined through detailed phylogenetic and structural analysis. In contrast, higher eukaryote MRP RNA structure models are based on comparative sequence analysis of only five sequences and limited probing data. Detailed structural analysis of the Homo sapiens MRP RNA, entailing enzymatic and chemical probing, is reported. The data are consistent with the phylogenetic secondary structure model and demonstrate unequivocally that higher eukaryote MRP RNA structure differs significantly from that reported for Saccharomycetaceae species. Neither model can account for all of the known MRP RNAs and we thus propose the evolution of at least two subsets of RNase MRP secondary structure, differing predominantly in the predicted specificity domain.

Algorithms↗
PubMed2006

RraA rescues Escherichia coli cells over-producing RNase E from growth arrest by modulating the ribonucleolytic activity.

RraA is an evolutionary conserved protein inhibitor of RNase E, which catalyzes the initial step in the decay and processing of numerous RNAs in Escherichia coli and forms the core component of the degradosome, a large protein complex involved in RNA metabolism. Here, we report that co-expression of RraA reduces the ribonucleolytic activity in cells over-producing RNase E and consequently rescues these cells from growth arrest. These findings suggest that inability of cells over-producing RNase E to normally grow results from increased cellular ribonucleolytic activity and RraA is able to effectively modulate the catalytic activity of RNase E in vivo.

Catalysis↗
PubMed2005

Adaptation to carbon starvation: RNase III ensures normal expression levels of bolA1p mRNA and sigma(S).

bolA is a sigma(S)-dependent Escherichia coli morphogene involved in the general cellular adaptation to stress and cell division. In this report it is shown that endoribonuclease RNase III acts as a post-transcriptional modulator of bolA expression under carbon starvation conditions. Unexpectedly RNase III positively regulates bolA1p mRNA levels and stabilities. This effect is also observed when sulA, bfr, uspA and uspB transcripts were analyzed. RNase III is furthermore shown to be necessary for the normal expression of sigma(S), ensuring normal levels of rpoS mRNA and sigma(S) protein under glucose starvation. Since sigma(S) controls a complex regulon of stress-response genes, RNase III is proposed as possible modulator of bacterial cell response to stress.

Adaptation, Biological↗
PubMed2004

Single processing center models for human Dicer and bacterial RNase III.

Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21 nt small interfering RNAs (siRNAs) during RNA interference, and excises microRNAs from precursor hairpins. Dicer contains two domains related to the bacterial dsRNA-specific endonuclease, RNase III, which is known to function as a homodimer. Based on an X-ray structure of the Aquifex aeolicus RNase III, models of the enzyme interaction with dsRNA, and its cleavage at two composite catalytic centers, have been proposed. We have generated mutations in human Dicer and Escherichia coli RNase III residues implicated in the catalysis, and studied their effect on RNA processing. Our results indicate that both enzymes have only one processing center, containing two RNA cleavage sites and generating products with 2 nt 3' overhangs. Based on these and other data, we propose that Dicer functions through intramolecular dimerization of its two RNase III domains, assisted by the flanking RNA binding domains, PAZ and dsRBD.

Amino Acid Sequence↗
PubMed2004

Studies of the RNA degradosome-organizing domain of the Escherichia coli ribonuclease RNase E.

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.

Amino Acid Sequence↗
PubMed2006

RNases and nucleases in embryos and endosperms from naturally aged wheat seeds stored in different conditions.

Temperature and moisture content are particularly important factors influencing the longevity of seeds, and therefore the ageing of seeds is closely tied to storage conditions. The ageing process is characterised by many physiological and biochemical changes: membranes tend to leak, enzymes lose catalytic activity, and chromosomes accumulate mutations. Since viability loss is also associated with the breakdown of nucleic acids, the aim of the study was to determine whether the damage induced by ageing could be associated with changes in the activity of RNases and nucleases in embryos and endosperms of differently stored wheat seeds. In order to better characterise seed conditions, the damage to membranes during seed ageing was evaluated by measuring the conductivity of the soaking solution during imbibition, and by using the Evans Blue colorant; lipid peroxidation was also recorded. RNases and nucleases were studied by SDS-PAGE and activity staining. Ageing of seeds stored in a dry state involved a progressive loss of membrane integrity, which increased with the degree of ageing, while lipid peroxidation remained unchanged. Changes in nucleolytic enzyme activity were recorded in embryos: a decrease in RNases and an increase in nucleases. In the endosperm compartment there were no significant differences in ribonuclease and nuclease patterns during seed ageing. Moreover, neutral RNases were absent in endosperms of dry seeds and were activated following imbibition. Present studies reveal that embryos and endosperms have different enzymatic patterns, thus highlighting that the two seed compartments age independently. A different nucleolytic pattern was present in seeds of comparable viability and membrane damage, which were stored differently, and nuclease metabolism was subject to regulation according to both ageing and the length of the storage period.

Deoxyribonucleases↗
PubMed2006

Structural basis for processivity and single-strand specificity of RNase II.

RNase II is a member of the widely distributed RNR family of exoribonucleases, which are highly processive 3'-->5' hydrolytic enzymes that play an important role in mRNA decay. Here, we report the crystal structure of E. coli RNase II, which reveals an architecture reminiscent of the RNA exosome. Three RNA-binding domains come together to form a clamp-like assembly, which can only accommodate single-stranded RNA. This leads into a narrow, basic channel that ends at the putative catalytic center that is completely enclosed within the body of the protein. The putative path for RNA agrees well with biochemical data indicating that a 3' single strand overhang of 7-10 nt is necessary for binding and hydrolysis by RNase II. The presence of the clamp and the narrow channel provides an explanation for the processivity of RNase II and for why its action is limited to single-stranded RNA.

Binding Sites↗
PubMed2006

RNase P: interface of the RNA and protein worlds.

Ribonuclease P (RNase P) is an endonuclease involved in processing tRNA. It contains both RNA and protein subunits and occurs in all three domains of life: namely, Archaea, Bacteria and Eukarya. The RNase P RNA subunits from bacteria and some archaea are catalytically active in vitro, whereas those from eukaryotes and most archaea require protein subunits for activity. RNase P has been characterized biochemically and genetically in several systems, and detailed structural information is emerging for both RNA and protein subunits from phylogenetically diverse organisms. In vitro reconstitution of activity is providing insight into the role of proteins in the RNase P holoenzyme. Together, these findings are beginning to impart an understanding of the coevolution of the RNA and protein worlds.

Animals↗
PubMed2002

3'-end processing of precursor M1 RNA by the N-terminal half of RNase E.

M1 RNA, the catalytic component of Escherichia coli RNase P, is derived from the 3'-end processing of precursor M1 RNA, a major transcript of the rnpB gene. In this study, we investigated the mechanism of 3'-end processing of M1 RNA using the recombinant N-terminal half RNase E. The cleavage site preference of RNase E differed from that of the 40% ammonium sulfate precipitate (ASP-40), a partially purified cell extract containing processing activity. However, the addition of a trace amount of ASP-40 changed the cleavage site preference of RNase E to that of ASP-40 suggesting the involvement of a soluble factor in cleavage site preference.

Base Sequence↗