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At least 109 records · Page 6Linked to original sources
PubMed1984

Location of RNase and RNase inhibitor on free cytoplasmic mRNA-protein particles from human placenta.

Free cytoplasmic mRNPs were isolated from human placenta. An activity of RNase was associated with these particles but was mostly inhibited by a labile protein inhibitor. Both RNase and RNase inhibitor were extractable from mRNPs by 0.5 M KCl. The nature of the association of the RNase-RNase inhibitor complex with mRNPs makes it suitable as a putative system for control of expression and turnover of mRNPs and therefore of protein synthesis.

Cytoplasm↗
PubMed2003

Escherichia coli RNase E and RNase G cleave a Bacillus subtilis transcript at the same site in a structure-dependent manner.

The decay of Bacillus subtilis aprE leader- lacZ mRNA was examined in Escherichia coli wild-type and in mutants deficient in RNase E, RNase G, or both. Two versions of the mRNA were studied: the wild-type mRNA, which has a stem-loop at the 5' end, and a mutant mRNA, with a single-stranded 5' end. The half-life of both transcripts was determined by RNase E, the half-life of the mutant transcript being one-third of that of the wild-type transcript. RNase G cleaved both transcripts at a site within an AU-rich sequence in the stem-loop region, but cleavage was much more efficient when the stem-loop was destabilized. RNase E cleaved at the same site, but less efficiently and only in the mutant transcript.

Bacillus subtilis↗
PubMed2003

Mutational analysis of the complex of human RNase inhibitor and human eosinophil-derived neurotoxin (RNase 2).

RNase inhibitor (RI) binds diverse proteins in the pancreatic RNase superfamily with extremely high avidity. Previous studies showed that tight binding of RNase A and angiogenin (Ang) is achieved primarily through interactions of hot spot residues in the 434-460 C-terminal segment of RI with the enzymatic active site; Asp435 of RI forms key hydrogen bonds with the catalytic lysine in both complexes, whereas the other contacts are largely distinctive. Here we have investigated the structural basis for recognition of a third ligand, eosinophil-derived neurotoxin (EDN), by single-site and multisite mutagenesis. Surprisingly, Ala replacement of Asp435 decreases affinity for EDN only by 14-fold, as compared to the several hundred-fold decreases with RNase A and Ang, and individual mutations of three other hot spot residues-Tyr434, Tyr437, and Ser460-have essentially no effect. Ala substitutions of nine additional residues, selected by examining a computational model of the RI.EDN complex, also have no marked impact. Overall, the losses in affinity for the single-residue variants examined account for only approximately 25% of the free energy of binding for the complex. However, multisite mutagenesis of RI reveals strong superadditivity of mutational effects, indicating that part of this shortfall reflects negative cooperativity. Replacement of Tyr434 together with Asp435 or Tyr437 increases K(i) by 540- and 290-fold, respectively. Thus, the C-terminal region of RI again plays an important role in ligand recognition, although probably smaller than for binding RNase A and Ang. Simultaneous substitutions of three neighboring tryptophans (261, 263, and 318) on RI attenuate affinity even more dramatically (by 4900-fold), indicating that the interactions of this RI region also contribute a considerable amount of the binding energy for the EDN complex. These findings highlight the potential importance of cooperativity in protein-protein interactions and the consequent limitations of single-site mutagenesis for assessing interface energetics.

Alanine↗
PubMed1989

Deoxynucleotide-containing oligoribonucleotide duplexes: stability and susceptibility to RNase V1 and RNase H.

Oligoribonucleotide duplexes containing one to four 2'-deoxynucleotide residues were used as substrates for ribonuclease V1 and RNase H. Either deoxyadenosine and/or deoxythymidine were incorporated into the duplex, 5'GGCCGGAUCCGCGC3'-5'GCGCGGAUCCGGCC3' by substitution of the appropriate deoxynucleoside triphosphate into a transcription reaction with T7 RNA polymerase. The melting temperature, Tm, of the duplex (1.8 microM in strands in 50 mM NaCl) containing only ribonucleotides was 79.9 degrees C. Substitution of deoxyadenosine in both strands of the duplex lowered the Tm by 2.4 degrees C. Substitution of deoxythymidine had no measurable effect on the Tm. Comparison of RNase V1 digestion patterns of fully ribonucleotide and deoxy-substituted duplexes suggest that any distortion is localized to the site of the substitution. An oligoribonucleotide containing two deoxy residues directs specific cleavage of RNA by E. coli RNase H. Structural requirements for cleavage are proposed for RNase V1 and RNase H.

Base Sequence↗
PubMed2005

Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site.

Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5' termini by cleaving tRNA precursors immediately 5' of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G+1C+72 base pair at the end of the aminoacyl acceptor-stem whereas in tRNA(Gln) G+1C+72 is replaced with U+1A+72. Here, we investigated RNase P RNA-mediated cleavage as a function of having G+1C+72 versus U+1A+72 in various substrate backgrounds, two full-size tRNA precursors (pre-tRNA(Gln) and pre-tRNA(Tyr)Su3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G+1C+72 with U+1A+72 influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNA(Gln) and pre-tRNA(Tyr)Su3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg2+ binding, most likely in its vicinity.

Base Pairing↗
PubMed1989

The difference spectra of bovine kidney RNase K2 induced upon binding with nucleotides markedly differ from those of bovine pancreatic RNase.

The difference spectra obtained upon the addition of nucleotides to bovine kidney RNase, which shows 40% sequence homology with bovine pancreatic RNase, are markedly different from those of bovine pancreatic RNase. As one of the factors which possibly contribute to this difference, we examined the effect of the substitution of Phe120 in bovine pancreatic RNase by Leu in RNase K2 on the difference spectra.

Amino Acid Sequence↗
PubMed2005

Opposite roles of the dmd gene in the control of RNase E and RNase LS activities.

When the dmd gene of bacteriophage T4 is defective, expression of middle genes starts normally but drops abruptly. However, the residual expression of middle genes at late stages continues at a higher rate in cells infected with a dmd mutant than with the wild type. In order to understand the complex effects of the dmd gene, we followed changes in the quantity of mRNA from a middle gene, uvsY. The uvsY mRNA was degraded rapidly by RNase LS at middle stages but stabilized at late stages, suggesting that RNase LS targets middle-gene mRNAs only at middle stages. Furthermore, another RNase targeting middle mRNAs at late stages is also suggested to be inactivated when dmd is mutated. We found that RNase E was involved in the degradation of uvsY mRNA. Judging from the processing of gene-32 mRNA, RNase E activity declines after the beginning of the middle stage when dmd is defective.

Bacteriophage T4↗
PubMed1999

The role of Escherichia coli RNase E and RNase III in the processing of the citQRP operon mRNA from Lactococcus lactis biovar diacetylactis.

Citrate transport in Lactococcus lactis biovar diacetylactis (L. diacetylactis) is catalyzed by citrate permease P (CitP), which is encoded by the plasmidic citP gene. Two partial overlapping open reading frames citQ and citR are located upstream of citP. These two genes, together with citP, constitute the citQRPoperon. In this report it was shown that in L. diacetylactis and Escherichia coli, cit mRNA is subject to the same specific cleavages at a complex secondary structure which includes the central region of citQ and the 5'-end of citR. The role of ribonucleases in the fate of the cit mRNA processing was investigated in E. coli RNase mutant strains. The results obtained indicate that both endoribonucleases RNase E and RNase III are involved in the generation of mRNA processed species. RNase E is responsible for the major cleavages detected within citQ and upstream of citR, whereas RNase III cleaves citR within its ribosomal binding site. Preliminary results indicate the existence of a RNaselll-like enzyme in L. diacetylactis. Based on these results, a model for the role of cit mRNA processing in the expression of citP is presented.

Bacterial Proteins↗
PubMed1985

Characterization of an RNase P activity from HeLa cell mitochondria. Comparison with the cytosol RNase P activity.

A ribonuclease P-like activity was partially purified from HeLa cell mitochondria by DEAE-cellulose and octyl-Sepharose chromatography. RNase P-like activity can be quantitatively recovered from intact mitochondrial preparations treated with micrococcal nuclease, strongly suggesting that the enzyme is localized within the organelles. Mitochondrial RNase P (mtRNase P) cleaves the precursor to Escherichia coli suppressor tRNATyr at the same site as E. coli RNase P, producing the mature 5'-end of tRNATyr. The sensitivity of mtRNase P to pretreatment with nucleases or Pronase indicates that the enzyme has essential RNA and protein components. Although the ionic requirements of mtRNase P are similar to those of the RNase P activity isolated from the post-mitochondrial cytosol fraction, the chromatographic properties of mtRNase P are distinct. Mitochondrial RNase P is probably a part of the mitochondrial RNA processing machinery of mammalian mitochondria, being responsible for the endonucleolytic cleavage of the RNA transcripts at the 5'-side of the tRNA sequences.

Adenosine Triphosphate↗
PubMed1995

HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA.

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA.

Base Sequence↗
PubMed2001

RNase activity of a DNA minor groove binder with a minimalist catalytic motif from RNase A.

Imidazole and compounds containing imidazole residues have been shown to cleave RNA in an RNase A-mimicking manner. Di-imidazole lexitropsin is a compound which is derived from the polyamide drugs distamycin and netropsin essentially by the replacement of two pyrrole heterocycles with N-methyl-imidazole residues. This enables it to bind to the minor groove of B-DNA in a sequence-specific manner. We demonstrate here that this lexitropsin derivative has RNA cleavage activity, as tested on model RNAs. Optimal cleavage conditions and cleavage specificity resemble those known from other imidazole conjugates and are thus consistent with an RNase A type cleavage mechanism. The optimum concentration of the compound for cleavage is similar to previously investigated imidazole-based RNase mimics. As a whole new class of chemical compounds capable of interacting with nucleic acids through extensive hydrogen bonding, these imidazole containing compounds constitute promising scaffolds and ligands, for the construction of novel RNase mimics with high affinity.

Animals↗
PubMed1984

Function of RNase H in DNA replication revealed by RNase H defective mutants of Escherichia coli.

Escherichia coli rnh mutants were isolated using localized mutagenesis and selective measurements of RNase H activity in mutagenized cell extracts with [3H]poly(rC) X poly(dG) as substrate. RNase H activity in extracts of one mutant, ON152 (rnh-91), was undetectable (less than 0.05% of that of wild-type cells). This mutant formed small colonies at 43 degrees C. At this temperature, accumulation of nascent fragments was more prominent in the rnh-91 X polA4113 double mutant than in the polA4113 mutant; however, no accumulation was found in the rnh single mutant at 43 degrees C. Unlike the 1-3 nucleotide primer RNA found on nascent fragments of polA4113 cells, primers from the rnh-91 X polA4113 cells ranged from one to about ten bases. These results suggest that the 5' leads to 3' exonuclease activity of DNA polymerase I plays a major role in removal of primer RNA and that RNase H functions in an auxiliary role, excising the 5'-portion of longer primers. The rnh mutant supports replication of ColE1-type plasmids. A possible mechanism of replication of such plasmids in rnh mutants and a role of RNase H in the initiation of chromosomal replication are discussed.

Coliphages↗
PubMed1984

RNase H-defective mutants of Escherichia coli: a possible discriminatory role of RNase H in initiation of DNA replication.

Mutants of Escherichia coli completely deficient in RNase H activity were isolated by inserting transposon Tn3 into the structural gene for RNase H, rnh, and its promoter. These rnh- mutants exhibited the following phenotypes; (1) the mutants grew fairly normally, (2) rnh- cells could be transformed with ColE1 derivative plasmids, pBR322 and pML21, though the plasmids were relatively unstable, under non selective conditions, (3) rnh- mutations partially suppressed the temperature-sensitive phenotype of plasmid pSC301, a DNA replication initiation mutant derived from pSC101, (4) rnh- mutations suppressed the temperature-sensitive growth character of dnaAts mutant, (5) rnh- cells showed continued DNA synthesis in the presence of chloramphenicol (stable DNA replication). Based on these findings we propose a model for a role of RNase H in the initiation of chromosomal DNA replication. We suggest that two types of RNA primers for initiation of DNA replication are synthesized in a dnaA/oriC-dependent and -independent manner and that only the dnaA/oriC-dependent primer is involved in the normal DNA replication since the dnaA/oriC independent primer is selectively degraded by RNase H.

DNA Replication↗
PubMed1988

Alpha-anomeric DNA: beta-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase.

Nuclease-resistant alpha-anomeric DNA:beta-RNA hybrids are inhibitors of Escherichia coli RNase H, and Drosophila embryo RNase H. RNase H activities were measured by polyacrylamide gel electrophoresis, employing a short substrate, (A)12:d[G-G-(T)12-G-G], or by acid-solubility techniques, using a long substrate, poly(A):poly(dT). Strand exchanges which could be responsible for the observed inhibition have been ruled out by S1 nuclease experiments and by using inhibitors which do not allow strand exchange. Our results suggest that RNase H, for which DNA:RNA duplexes are the natural substrates, binds to non-physiological alpha-DNA:RNA hybrids and is consequently inhibited. These hybrids also inhibit the RNA-dependent DNA polymerase activity of M-MLV reverse transcriptase, therefore appearing as potential inhibitors of at least two reverse transcriptase activities. However, the inhibitory effect of these hybrids with respect to M-MLV reverse transcriptase is also observed with the single-stranded alpha-DNA itself. Unexpectedly, polymerase activity is highly stimulated by alpha-oligos, analogous in their sequence to the beta primer used at a concentration unable to generate a detectable synthesis. These results suggest that the inhibition of reverse transcriptase activity with the alpha:beta may occur at different levels.

Animals↗
PubMedDate not supplied

Selective mRNA degradation by antisense oligonucleotide-2,5A chimeras: involvement of RNase H and RNase L.

Antisense oligonucleotides (ON) allow the specific control of gene expression and phosphorothioate derivatives are currently being evaluated for possible clinical applications. Numerous second generation ON analogues with improved pharmacological properties have been described. Most of them, however, do not recruit RNase H, which is known to increase ON potency by eliciting the specific degradation of the target RNA. Silverman, Torrence and colleagues have conjugated 2,5A to natural antisense ON and demonstrated the preferential cleavage of a target RNA in cell-free and intact cell experiments. We have established for the first time that RNase H-incompetent ON, viz. alpha-anomeric ON analogues, can be converted into sequence-specific nucleases upon conjugation to 2,5A. The use of alpha-ON- and beta-ON-2,5A chimeras has allowed us to delineate the part played by RNase H and RNase L in target RNA degradation and translation arrest. Finally, the present studies have revealed limitations which are encountered in the choice of a suitable target for such ON-2,5A chimeras.

Animals↗
PubMed1986

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements.

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.

Base Composition↗
PubMed1996

Effects of protein RNase inhibitor and substrate on the quaternary structures of bovine seminal RNase.

The effect of the protein RNase inhibitor (PRI) on the activity of bovine seminal RNase (BS-RNase) was investigated using the isolated quaternary forms, MxM and M=M, of the enzyme reported earlier [Piccoli, R., et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1870-1874]. We found that the inhibitor does not interact with the intact isolated forms but has dramatic, differential effects on the two forms when the assays are performed under reducing conditions. These conditions, which are essential for full activity of the inhibitor, and are typical of its cytosolic localization, also promote monomerization of the M=M form, while under identical conditions the MxM form becomes a noncovalent dimer (NCD). The sensitivity of BS-RNase or that of the isolated quaternary forms under reducing conditions thus appears to be related to differential monomerization of the two forms of the enzyme; monomer being sensitive to PRI. The present study also shows that the interconversion between the two forms in equilibrium occurs at much higher rates in a reducing environment and that PRI further affects the interconversion and alters the equilibrium favoring monomerization of the protein. An opposite effect on the equilibrium between the forms is played by the substrate, which is found to stabilize the NCD form of the protein with a shift in the equilibrium between the two forms towards the dimer. These results are analyzed in the light of the antitumor action of the enzyme which is exerted in the cytosol, i.e., in the compartment housing the PRI and the ribosomal RNA, the molecular target of the enzyme.

Animals↗
PubMed2003

An oligopeptide containing the C-terminal sequence of RNase a has a potent RNase a binding property.

We demonstrate that an oligopeptide containing the C-terminal sequence of RNase A binds to RNase A in a stoichiometric and site-specific manner. Our observations are consistent with the interaction found in the major domain-swapped RNase A dimer, so that the peptide binding may be promoted through the swapping with the C-terminal beta-sheet of RNase A. Because the design of a protein-binding peptide is much simpler than other methods such as the combinatorial method, we propose that investigation using an oligopeptide may be of general application to domain swapping in proteins as well as for the development of an oligopeptide tool that specifically binds to a target protein.

Amino Acid Sequence↗