Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNAse”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed1994

An active recombinant p15 RNase H domain is functionally distinct from the RNase H domain associated with human immunodeficiency virus type 1 reverse transcriptase.

An active p15 RNase H domain, consisting of amino acids 427-560 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and a genetically engineered penta-histidine N-terminal affinity tag, was expressed in Escherichia coli and purified to apparent homogeneity by immobilized metal affinity chromatography. The purified p15 RNase H domain exhibited no substrate preference for [3H]poly(rG).poly(dC) compared to [3H]poly(rA).poly(dT), in contrast with the HIV-1 RT-associated RNase H, which showed a 30-fold preference for the former substrate. Unlike the HIV-1 RT-associated RNase H, when challenged with unlabeled substrate, the recombinant p15 RNase H domain was relatively nonprocessive in RNA degradative activity of the [3H]poly(rA).poly(dT) duplex. Kinetic studies using p15 RNase H showed substrate inhibition with an apparent K(i) value of 0.12 micron for the [3H]poly(rA).poly(dT) hybrid. Substrate inhibition was not observed for the HIV-1 RT-associated RNase H. The results show that the isolated p15 HIV-1 RNase H domain is functionally distinct from the recombinant HIV-1 RT-associated RNase H.

Amino Acid Sequence↗
PubMed1995

Defects in primer-template binding, processive DNA synthesis, and RNase H activity associated with chimeric reverse transcriptases having the murine leukemia virus polymerase domain joined to Escherichia coli RNase H.

The RNase H domain of murine leukemia virus (MuLV) reverse transcriptase (RT) was replaced with Escherichia coli RNase H, and the effect on RNase H activity and processive DNA synthesis was studied, using RNA-DNA hybrids containing sequences from the MuLV polypurine tract (PPT). Two chimeric RTs, having the entire polymerase domain or all but the last 19 amino acids, were expressed. In both cases, these RTs made multiple cuts in PPT-containing substrates, whereas wild-type cleavages occurred primarily at sites consistent with the distance between the polymerase and RNase H active sites. Primer extension assays performed with the chimeric RTs, an RNase H-minus RT, and wild-type showed that the presence of a wild-type viral RNase H domain facilitates processive DNA synthesis. When wild-type RT was bound to primer-template, two retarded bands could be detected in band-shift assays. In the absence of primer extension, a high proportion of enzyme-bound primer-template was associated with the faster-migrating band, whereas with DNA synthesis, more of the bound radioactivity was in the super-shifted complex. This suggests that the super-shifted complex contains the active form of RT. The mutant RTs were deficient in formation of this complex, but the chimeric RTs were somewhat less defective than the RNase H-minus mutant. Our results demonstrate that in the wild-type enzyme, the RNase H domain is required to stabilize the interaction between RT and primer-template.

Base Sequence↗
PubMed2002

RNase G complementation of rne null mutation identifies functional interrelationships with RNase E in Escherichia coli.

The Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have sequence similarity and broadly similar sequence specificity. Whereas the absence of Rne normally is lethal, we show here that E. coli bacteria that lack the rne gene can be made viable by overexpression of Rng. Rng-complemented cells accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase P (i.e. M1 RNA), indicating that normal processing of these Rne-cleaved RNAs was not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated from precursors by RNase G cleavage in vitro. Using DNA microarrays containing 4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose steady-state level was affected by Rne, Rng or the N-terminal catalytic domain of RNase E. Most transcript species affected by RNase E deficiency were also elevated in an rne deletion mutant complemented by Rng. However, approximately 100 mRNAs that accumulated in Rne-deficient cells were decreased by rng-complemention, thus identifying targets whose processing or degradation may be the basis for RNase E essentiality. Remarkably prominent in this group were mRNAs implicated in energy-generating pathways or in the synthesis or degradation of macromolecules.

Base Sequence↗
PubMed2003

RNase G of Escherichia coli exhibits only limited functional overlap with its essential homologue, RNase E.

RNase G (rng) is an E. coli endoribonuclease that is homologous to the catalytic domain of RNase E (rne), an essential protein that is a major participant in tRNA maturation, mRNA decay, rRNA processing and M1 RNA processing. We demonstrate here that whereas RNase G inefficiently participates in the degradation of mRNAs and the processing of 9S rRNA, it is not involved in either tRNA or M1 RNA processing. This conclusion is supported by the fact that inactivation of RNase G alone does not affect 9S rRNA processing and only leads to minor changes in mRNA half-lives. However, in rng rne double mutants mRNA decay and 9S rRNA processing are more defective than in either single mutant. Conversely, increasing RNase G levels in an rne-1 rng::cat double mutant, proportionally increased the extent of 9S rRNA processing and decreased the half-lives of specific mRNAs. In contrast, variations in the amount of RNase G did not alter tRNA processing under any circumstances. Thus, the failure of RNase G to complement rne mutations, even when overproduced at high levels, apparently results from its inability to substitute for RNase E in the maturation of tRNAs.

Alleles↗
PubMed1990

Isolation and characterization of a second RNase H (RNase HII) of Escherichia coli K-12 encoded by the rnhB gene.

An additional RNase H (EC 3.1.26.4), RNase HII, has been isolated from Escherichia coli K-12. By screening a library of E. coli DNA for clones that suppressed RNase H deficiency of an E. coli rnh mutant, a clone was obtained that produced a protein with RNase H activity. The overexpressed RNase HIII protein in E. coli was purified to near homogeneity and exhibited a strong preference for the ribonucleotide moiety of RNA-DNA hybrid as substrate. The terminal 11 amino acids were determined and were identical to those predicted from the nucleotide sequence. The rnhB gene, which encodes RNase HII, was distinct from rnhA by its map position (4.5 min on E. coli genetic map, between lpxB and dnaE) and by the lack of significant amino acid sequence similarity. The presence of a second RNase H in E. coli indicates that multiple RNase H genes per genome is a general feature of a general feature of a wide variety of organisms.

Amino Acid Sequence↗
PubMed2001

Characterization of Rny1, the Saccharomyces cerevisiae member of the T2 RNase family of RNases: unexpected functions for ancient enzymes?

The T(2) family of nonspecific endoribonucleases (EC ) is a widespread family of RNases found in every organism examined thus far. Most T(2) enzymes are secretory RNases and therefore are found extracellularly or in compartments of the endomembrane system that would minimize their contact with cellular RNA. Although the biological functions of various T(2) RNases have been postulated on the basis of enzyme location or gene expression patterns, the cellular roles of these enzymes are generally unknown. In the present work, we characterized Rny1, the only T(2) RNase in Saccharomyces cerevisiae. Rny1 was found to be an active, secreted RNase whose gene expression is controlled by heat shock and osmotic stress. Inactivation of RNY1 leads to unusually large cells that are temperature-sensitive for growth. These phenotypes can be complemented not only by RNY1 but also by both structurally related and unrelated secretory RNases. Additionally, the complementation depends on RNase activity. When coupled with a recent report on the effect of specific RNAs on membrane permeability [Khvorova, A., Kwak, Y-G., Tamkun, M., Majerfeld, I. & Yarus, M. (1999) Proc. Natl. Acad. Sci. USA 96, 10649-10654], our work suggests an unexpected role for Rny1 and possibly other secretory RNases. These enzymes may regulate membrane permeability or stability, a hypothesis that could present an alternative perspective for understanding their functions.

Endoribonucleases↗
PubMed2005

The RNase Z homologue encoded by Escherichia coli elaC gene is RNase BN.

In eukaryotes, archaea, and in some eubacteria, removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, whose function is under active study. We have overexpressed and purified to homogeneity His-tagged ElaC and show here that it is, in fact, the previously described enzyme, RNase BN. Thus, purified ElaC displays structural and catalytic properties identical to those ascribed to RNase BN. In addition, an elaC mutant strain behaves identically to a known RNase BN- strain, CAN. Finally, we show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity. These data correct a previous misassignment for the gene encoding RNase BN. Based on the fact that the original RNase BN mutation has now been identified, we propose that the elaC gene be renamed rbn.

Bacteriophages↗
PubMed2006

Ribonucleases in HIV type 1 inhibition: effect of recombinant RNases on infection of primary T cells and immune activation-induced RNase gene and protein expression.

Ribonucleases (RNases) have therapeutic potential against cancer and viral diseases and have been reported to inhibit replication of the human immunodeficiency virus type 1 (HIV-1) in chronically infected cell lines. The ribonuclease eosinophil-derived neurotoxin (EDN) is responsible for the anti-HIV-1 activity of a soluble factor produced in response to human alloantigens (ASF). Four recombinant RNases (EDN; a four amino acid extension of the N-terminus EDN, -4EDN; RNase A; and angiogenin) were tested for inhibition of HIV-1 replication in PHA blasts. All RNases showed anti-HIV-1 activity, irrespective of whether the RNases were added before, during, or 2 h after infection. Polyclonal antibodies against the four RNases blocked the antiviral activity. ASF inhibited HIV-1 replication in vitro if added up to 4 h after infection. We demonstrated that allostimulation induced EDN, RNase A, and angiogenin mRNA expression in peripheral blood mononuclear cells (PBMCs), although only EDN protein was detected. We identified monocytes and dendritic cells, but not macrophages or T cells, as EDN-producing cells. These findings raise the possibilities that multiple naturally occurring RNases may contribute to protection against HIV-1 infection and could be considered for utilization in HIV-1 therapy.

Analysis of Variance↗
PubMed1999

RNase G (CafA protein) and RNase E are both required for the 5' maturation of 16S ribosomal RNA.

In Escherichia coli, rRNA operons are transcribed as 30S precursor molecules that must be extensively processed to generate mature 16S, 23S and 5S rRNA. While it is known that RNase III cleaves the primary transcript to separate the individual rRNAs, there is little information about the secondary processing reactions needed to form their mature 3' and 5' termini. We have now found that inactivation of the endoribonuclease RNase E slows down in vivo maturation of 16S RNA from the 17S RNase III cleavage product. Moreover, in the absence of CafA protein, a homolog of RNase E, formation of 16S RNA also slows down, but in this case a 16.3S intermediate accumulates. When both RNase E and CafA are inactivated, 5' maturation of 16S rRNA is completely blocked. In contrast, 3' maturation is essentially unaffected. The 5' unprocessed precursor that accumulates in the double mutant can be assembled into 30S and 70S ribosomes. Precursors also can be processed in vitro by RNase E and CafA. These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5' end of 16S rRNA, and that CafA protein is a new ribonuclease. We propose that it be renamed RNase G.

Bacterial Proteins↗
PubMed2003

Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors.

Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.

Binding Sites↗
PubMed2005

Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB.

Previous work has demonstrated that iron-dependent variations in the steady-state concentration and translatability of sodB mRNA are modulated by the small regulatory RNA RyhB, the RNA chaperone Hfq and RNase E. In agreement with the proposed role of RNase E, we found that the decay of sodB mRNA is retarded upon inactivation of RNase E in vivo, and that the enzyme cleaves within the sodB 5'-untranslated region (5'-UTR) in vitro, thereby removing the 5' stem-loop structure that facilitates Hfq and ribosome binding. Moreover, RNase E cleavage can also occur at a cryptic site that becomes available upon sodB 5'-UTR/RyhB base pairing. We show that while playing an important role in facilitating the interaction of RyhB with sodB mRNA, Hfq is not tightly retained by the RyhB-sodB mRNA complex and can be released from it through interaction with other RNAs added in trans. Unlike turnover of sodB mRNA, RyhB decay in vivo is mainly dependent on RNase III, and its cleavage by RNase III in vitro is facilitated upon base pairing with the sodB 5'-UTR. These data are discussed in terms of a model, which accounts for the observed roles of RNase E and RNase III in sodB mRNA turnover.

5' Untranslated Regions↗
PubMed1982

Fluorescence titrations of residue 59 and tyrosine in Kyn 59-RNase T1 and NFK 59-RNase T1.

Fluorescence titrations of kynurenine and tyrosine in Kyn 59-RNase T1 and NFK 59-RNase T1 were carried out by monitoring protein fluorescence through a pH change from 1.5 to 10.5. In the titration of kynurenine fluorescence at 455 nm, a few small but distinct quenching events occurred between pH 3.5 and 9.5. Three ionizable groups were found to be responsible for the individual steps of quenching observed. These groups are Glu 58 with pKa 4.6, His 40 or 92 with pKa 7.8 and Lys 41 with pKa 8.7. From this result, a subtle conformational change associated with the proton dissociation equilibria of Glu 58 and His 40 or 92 in the active site of Kyn 59-RNase T1 is suggested. The pH-titration behavior of tyrosine fluorescence in Kyn 59-RNase T1 was different from that of kynurenine fluorescence. Two acidic groups with pKa's 3.2 and 6.5 were detected as perturbants. In NFK 59-RNase T1, both N'-formylkynurenine and tyrosine showed almost the same fluorescence behavior during titration, which was characterized by two transitions between pH 3 and 8 in each titration curve. Two ionizable groups with pKa's 3.7-3.8 and 6.7-6.8 were determined. The role of the latter ionizable group is discussed in relation to the enzyme function of RNase T1. From the close similarity in structure and function between Kyn 59-RNase T1 and RNase T1, it is suggested that the same mechanism of conformational change linked to the ionization states of Glu 58 and His 40 or 92 exists in the native protein too.

Hydrogen-Ion Concentration↗
PubMed1994

The POP1 gene encodes a protein component common to the RNase MRP and RNase P ribonucleoproteins.

Two forms of the yeast 5.8S rRNA are generated from a large precursor by distinct processing pathways. Cleavage at site A3 is required for synthesis of the major, short form, designated 5.8S(S), but not for synthesis of the long form, 5.8S(L). To identify components required for A3 cleavage, a bank of temperature-sensitive lethal mutants was screened for those with a reduced ratio of 5.8S(S):5.8S(L). The pop1-1 mutation (for processing of precursor RNAs) shows this phenotype and also inhibits A3 cleavage. The pre-rRNA processing defect of pop1-1 strains is similar to that reported for mutations in the RNA component of RNase MRP; we show that a mutation in the RNase MRP RNA also inhibits cleavage at site A3. This is the first site shown to require RNase MRP for cleavage in vivo. The pop1-1 mutation also leads to a block in the processing of pre-tRNA that is identical to that reported for mutations in the RNA component of RNase P. The RNA components of both RNase MRP and RNase P are underaccumulated in pop1-1 strains at the nonpermissive temperature, and immunoprecipitation demonstrates that POP1p is a component of both ribonucleoproteins. The POP1 gene encodes a protein with a predicted molecular mass of 100.5 kD and is essential for viability. POP1p is the first protein component of the nuclear RNase P or RNase MRP for which the gene has been cloned.

Amino Acid Sequence↗
PubMed2005

On a salmon (Oncorhynchus [corrected] keta) liver RNase, belonging to RNase T2 family: primary structure and some properties.

A base-nonspecific and acid ribonuclease (RNase Ok2) was purified from the liver of a salmon (Oncorhnchus keta) to a homogeneous state by SDS-PAGE. The primary structure of RNase Ok2 was determined by protein chemistry and molecular cloning. The RNase Ok2 was a glycoprotein and consisted of 216 amino acid residues. Its molecular mass of protein moiety was 25,198, and its amino acid sequence showed that it belongs to the RNase T2 family of enzymes. The optimal pH of RNase Ok2 was around 5.5. The base preferences at the B1 and B2 sites were estimated from the rates of hydrolysis of 16 dinucleoside phosphates to be G>>A>U, C, and G>A>U>C respectively. In this enzyme, one of the three histidine residues which have been thought to be important for catalysis of RNase Rh, a typical RNase of this family of enzymes, His104 was replaced by tyrosine residue. Based on the results, the role of H104, which has been proposed to be a phosphate binding site with a substrate, was reconsidered, and we proposed a revised role of this His residue in the hydrolysis mechanism of RNase T2 family enzymes.

Amino Acid Sequence↗
PubMed2002

Identity of the RNase MRP- and RNase P-associated Th/To autoantigen.

OBJECTIVE: To characterize the molecular identity of the Th/To autoantigen, which is targeted by autoantibodies in scleroderma and which is associated with the human RNase MRP and RNase P ribonucleoprotein complexes. METHODS: Proteins immunoprecipitated by anti-Th/To+ patient antisera from biotinylated total HeLa cell extracts were analyzed by immunoblotting. The association of autoantigenic proteins with the RNase MRP complex was analyzed by reconstitution experiments and ultraviolet crosslinking. The reactivity of patient sera with all known RNase MRP/RNase P proteins was analyzed by immunoprecipitation of the individual recombinant proteins. RESULTS: The previously defined Th40 autoantigen appeared to be identical to the Rpp38 protein. Paradoxically, Rpp38 did not bind to the P3 domain of the RNase MRP RNA, as suggested by previously published data for Th40, and only half of the anti-Th/To+ sera contained anti-Rpp38 reactivity. Two other RNase MRP/RNase P subunits, Rpp20 and Rpp25, were found to interact with the P3 domain. The previously reported 40-kd species associated with this domain appeared to consist of Rpp20 and/or Rpp25 associated with a nuclease-resistant RNA fragment. Finally, we demonstrated that almost all tested anti-Th/To+ patient sera contained autoantibodies to Rpp25 and hPop1, indicating that these proteins harbor the most frequently targeted Th/To determinants. CONCLUSION: Our data unequivocally define the identity of the Th/To autoantigen and demonstrate that Th/To autoepitopes are found on several protein subunits of RNase MRP/RNase P.

Apoptosis Regulatory Proteins↗
PubMed1996

The evolution of the RNase P- and RNase MRP-associated RNAs: phylogenetic analysis and nucleotide substitution rate.

We report a detailed evolutionary study of the RNase P- and RNase MRP- associated RNAs. The analyses were performed on all the available complete sequences of RNase MRP (vertebrates, yeast, plant), nuclear RNase P (vertebrates, yeast), and mitochondrial RNase P (yeast) RNAs. For the first time the phylogenetic distance between these sequences and the nucleotide substitution rates have been quantitatively measured.The analyses were performed by considering the optimal multiple alignments obtained mostly by maximizing similarity between primary sequences. RNase P RNA and MRP RNA display evolutionary dynamics following the molecular clock. Both have similar rates and evolve about one order of magnitude faster than the corresponding small rRNA sequences which have been, so far, the most common gene markers used for phylogeny. However, small rRNAs evolve too slowly to solve close phylogenetic relationships such as those between mammals. The quicker rate of RNase P and MRP RNA allowed us to assess phylogenetic relationships between mammals and other vertebrate species and yeast strains. The phylogenetic data obtained with yeasts perfectly agree with those obtained by functional assays, thus demonstrating the potential offered by this approach for laboratory experiments.

Animals↗
PubMed2000

Polynucleotide phosphorylase, RNase II and RNase E play different roles in the in vivo modulation of polyadenylation in Escherichia coli.

Poly(A) tails in Escherichia coli are hypothesized to provide unstructured single-stranded substrates that facilitate the degradation of mRNAs by ribonucleases. Here, we have investigated the role that such nucleases play in modulating polyadenylation in vivo by measuring total poly(A) levels, polyadenylation of specific transcripts, growth rates and cell viabilities in strains containing various amounts of poly(A) polymerase I (PAP I), polynucleotide phosphorylase (PNPase), RNase II and RNase E. The results demonstrate that both PNPase and RNase II are directly involved in regulating total in vivo poly(A) levels. RNase II is primarily responsible for degrading poly(A) tails associated with 23S rRNA, whereas PNPase is more effective in modulating the polyadenylation of the lpp and 16S rRNA transcripts. In contrast, RNase E appears to affect poly(A) levels indirectly through the generation of new 3' termini that serve as substrates for PAP I. In addition, whereas excess PNPase suppresses polyadenylation by more than 70%, the toxicity associated with increased poly(A) levels is not reduced. Conversely, toxicity is significantly reduced in the presence of excess RNase II. Overproduction of RNase E leads to increased polyadenylation and no reduction in toxicity.

Endoribonucleases↗
PubMed2002

Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo.

Retroviral reverse transcriptases contain a DNA polymerase activity that can copy an RNA or DNA template and an RNase H activity that degrades the viral RNA genome during reverse transcription. RNase H makes both specific and nonspecific cleavages; specific cleavages are used to generate and remove the polypurine tract primer used for plus-strand DNA synthesis and to remove the tRNA primer used for minus-strand DNA synthesis. We generated mutations in an HIV-1-based vector to change amino acids in the RNase H domain that contact either the RNA and DNA strands. Some of these mutations affected the initiation of DNA synthesis, demonstrating an interdependence of the polymerase and RNase H activities of HIV-1 reverse transcription during viral DNA synthesis. The ends of the linear DNA form of the HIV-1 genome are defined by the specific RNase H cleavages that remove the plus- and minus-strand primers; these ends can be joined to form two-long-terminal repeat circles. Analysis of two-long-terminal repeat circle junctions showed that mutations in the RNase H domain affect the specificity of RNase H cleavage.

Base Sequence↗