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At least 343 records · Page 19Linked to original sources
PubMed2000

Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond.

BACKGROUND: The microscopic events of ribonuclease (RNase) catalyzed phosphoryl transfer reactions are still a matter of debate in which the contenders adhere to either the classical concerted acid-base mechanism or a more sequential triester-like mechanism. In the case of RNase A, small thio-effects of the nonbridging oxygens have been invoked in favor of the classical mechanism. However, the RNase T1 catalyzed transphosphorylation of phosphorothioate RNA is highly stereoselective. R(P) thio-substituted RNA is depolymerized 60000 times faster than S(P) thio-substituted RNA by this enzyme, whereas the uncatalyzed cleavage of both substrates occurs at comparable rates. We combined site-directed mutagenesis in the RNase active site and stereospecific thio-substitution of an RNA substrate to probe the intermolecular interactions of the enzyme with the nonbridging pro-S(P) oxygen that bring about this stereoselectivity of RNase T1. RESULTS: Thio-substitution of the nonbridging pro-S(P) oxygen in the substrate afflicts chemical turnover but not ground state binding whereas thio-substitution of the nonbridging pro-R(P) oxygen does not affect the kinetics of RNase T1. Site-directed mutagenesis of the catalytic base Glu58 impairs the enzyme's ability to discriminate both phosphorothioate diastereomers. Glu58Ala RNase T1 cleaves R(P) and S(P) phosphorothioate RNA with similar rates. The dependence of the pro-S(P) thio-effect on the presence of the Glu58 carboxylate evidences a strong rate-limiting interaction between the nonbridging pro-S(P) oxygen and the catalytic base Glu58 in the wild type enzyme. CONCLUSIONS: Based on these results, we put forward a new triester-like mechanism for the RNase T1 catalyzed reaction that involves a three-centered hydrogen bond between the 2'-OH group, the nonbridging pro-S(P) oxygen and one of the carboxylate oxygens of Glu58. This interaction allows nucleophilic attack on an activated phosphate to occur simultaneously with general base catalysis, ensuring concerted phosphoryl transfer via a triester-like mechanism.

Binding Sites↗
PubMed2000

RNase II removes the oligo(A) tails that destabilize the rpsO mRNA of Escherichia coli.

Polyadenylation controls mRNA stability in procaryotes, eucaryotes, and organelles. In bacteria, oligo(A) tails synthesized by poly(A) polymerase I are the targets of the 3'-to-5' exoribonucleases: polynucleotide phosphorylase and RNase II. Here we show that RNase II very efficiently removes the oligo(A) tails that can be used as binding sites by PNPase to start degradation of the rpsO mRNA. Both enzymes are impeded by the secondary structure of the transcription terminator at the 3' end of the mRNA. RNase II mostly generates tailless transcripts harboring 2 unpaired nt downstream of the transcription terminator hairpin, whereas PNPase releases molecules that exhibit a single-stranded stretch of 5-7 nt terminated by a tail of 3-5 As. The rpsO mRNAs whose oligo(A) tails have been removed by RNase II are more stable than oligoadenylated molecules that occur in strains deficient for RNase II. Moreover, the rpsO mRNA is stabilized when RNase II is overproduced. This modulation of mRNA stability by RNase II is only observed when poly(A) polymerase I is active. These in vivo data demonstrate that RNase II protects mRNAs ending by stable terminal hairpins, such as primary transcripts, from degradation by poly(A)-dependent ribonucleases.

Blotting, Northern↗
PubMed1999

Maturation of 23S ribosomal RNA requires the exoribonuclease RNase T.

Ribosomal RNAs are generally synthesized as long, primary transcripts that must be extensively processed to generate the mature, functional species. In Escherichia coli, it is known that the initial 30S precursor is cleaved during its synthesis by the endonuclease RNase III to generate precursors to the 16S, 23S, and 5S rRNAs. However, despite extensive study, the processes by which these intermediate products are converted to their mature forms are poorly understood. In this article, we describe the maturation of 23S rRNA. Based on Northern analysis of RNA isolated from a variety of mutant strains lacking one or multiple ribonucleases, we show that maturation of the 3' terminus requires the action of RNase T, an enzyme previously implicated in the end turnover of tRNA and in the maturation of small, stable RNAs. Although other exoribonucleases can participate in shortening the 3' end of the initial RNase III cleavage product, RNase T is required for removal of the last few residues. In the absence of RNase T, 23S rRNA products with extra 3' residues accumulate and are incorporated into ribosomes, with only small effects on cell growth. Purified RNase T accurately and efficiently converts these immature ribosomes to their mature forms in vitro, whereas free RNA is processed relatively poorly. In vivo, the processing defect at the 3' end has no effect on 5' maturation, indicating that the latter process proceeds independently. We also find that a portion of the 23S rRNA that accumulates in many RNase T- cells becomes polyadenylated because of the action of poly(A) polymerase I. The requirement for RNase T in 23S rRNA maturation is discussed in relation to a model in which only this enzyme, among the eight exoribonucleases present in E. coli, is able to efficiently remove nucleotides close to the double-stranded stem generated by the pairing of the 5' and 3' termini of most stable RNAs.

Blotting, Northern↗
PubMed2002

Dimeric and monomeric Bacillus subtilis RNase P holoenzyme in the absence and presence of pre-tRNA substrates.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that catalyzes the 5' maturation of tRNA precursors. The bacterial RNase P holoenzyme is composed of a large, catalytic RNA and a small protein. Our previous work showed that Bacillus subtilis RNase P forms a specific "dimer" that contains two RNase P RNA and two RNase P protein subunits in the absence of substrate. We investigated the equilibrium and the structure of the dimeric and the monomeric holoenzyme in the absence and presence of substrates by synchrotron small-angle X-ray scattering, 3' autolytic processing, and hydroxyl radical protection. In the absence of substrate, the dimer-monomer equilibrium is sensitive to monovalent ions and the total holoenzyme concentration. At 0.1 M NH4Cl, formation of the dimer is strongly favored, whereas at 0.8 M NH4Cl, the holoenzyme is a monomer. Primary hydroxyl radical protection in the dimer is located in the specificity domain, or domain I, of the RNase P RNA. The ES complex with a substrate containing a single tRNA is always monomeric. In contrast, the dominant ES complex with a substrate containing two tRNAs is dimeric at 0.1 M NH4Cl and monomeric at 0.8 M NH4Cl. Our results show that the B. subtilis holoenzyme can be a dimer and a monomer, and the fraction of the dimer is very sensitive to the environment. Under a variety of conditions, both the holoenzyme dimer and monomer can be present in significant amounts. Because the majority of tRNA genes are organized in large operons and because of the lack of RNase E in B. subtilis, a dimeric holoenzyme may be necessary to facilitate the processing of large precursor tRNA transcripts. Alternatively, the presence of two forms of the RNase P holoenzyme may be required for other yet unknown functions.

Bacillus subtilis↗
PubMed2006

Increase of RNase a N-terminus polarity or C-terminus apolarity changes the two domains' propensity to swap and form the two dimeric conformers of the protein.

Do the polarities of the N-terminus or the apolarity of the C-terminus of bovine RNase A influence the relative yields of its two 3D domain-swapped dimeric conformers, the N-dimer and C-dimer? We have addressed this question by substituting Ala-4 or Ala-5 with serine (A4S and A5S mutants) or Ser-123 with alanine (S123A mutant) through site-directed mutagenesis. Both the polarity of the N-terminus and the apolarity of the C-terminus of RNase A were, therefore, increased. CD spectra revealed no significant differences between the secondary structures of the mutants and native RNase A. According to thermal denaturation analyses, the A4S and A5S mutants are less stable, and the S123A mutant is more stable than wild type RNase A. By subjecting the mutants under mild or drastic denaturing conditions, side-by-side with native and recombinant RNase A, to a thermally induced oligomerization procedure, the following results were obtained. (i) The N-terminal mutants showed a higher propensity, with respect to the native protein, to form N-dimers under mild unfolding conditions. (ii) The C-terminal mutant showed a higher propensity to form the C-dimer under severely unfolding conditions. These results are discussed in light of the relative stabilities of the various RNase A species under different environmental conditions, and we conclude that the hydrophilic or hydrophobic character of the RNase N-terminus or C-terminus can be an important variable governing the oligomerization of RNase A and possibly other proteins through the 3D domain-swapping mechanism.

Alanine↗
PubMed1996

Glutathione-dependent pathways of refolding of RNase T1 by oxidation and disulfide isomerization: catalysis by protein disulfide isomerase.

Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase T1 (rd-RNase T1) and mixed disulfide between glutathione and reduced RNase T1 (GS-RNase T1) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ES-MS) time-course analysis. This permitted both the identification and quantitation of the population of intermediates present during the refolding process. Refolding experiments were performed in the presence of different absolute concentrations of glutathione species while keeping the redox potential fixed, in order to evaluate the effect of the glutathione concentration on the distribution of the refolding intermediates. All the analyses indicate a pathway of sequential reactions in the formation of native RNase T1 which occurs via the reiteration of two steps: (i) formation of a species containing both mixed disulfides with glutathione and free protein thiols, and (ii) formation of an intramolecular disulfide via thiol-disulfide interchange reaction between them. Refolding of rd-RNase T1 and GS-RNase T1 was also performed in the presence of protein disulfide isomerase (PDI). Addition of PDI led to a catalysis of each individual reaction of the entire process without altering the refolding pathway. Refolding reactions carried out at different absolute concentrations of glutathione proved that GSH and/or GSSG participate directly in the reaction catalyzed by PDI. On the basis of these experiments and previous results on the refolding of RNase A [Torella, C., Ruoppolo, M., Marino, G., & Pucci, P. (1994) FEBS Lett. 352, 301-306], a hypothesis of a general pathway for folding of S--S containing proteins is proposed.

Animals↗
PubMedDate not supplied

Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use.

The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.

Animals↗
PubMed1998

Senescence-induced RNases in tomato.

A main feature of leaf senescence is the hydrolysis of macromolecules by hydrolases of various types, and redistribution of released materials. We have initiated a study for the characterization of RNases involved in nucleic acid catabolism during senescence. Using a PCR-based cloning approach we isolated from tomato two senescence-induced RNase cDNA clones. Each of these cDNAs hybridized to a senescence-induced transcript in northern analysis. One RNase cDNA was identical to the tomato LX RNase while the second corresponded to the LE RNase. Both LX and LE RNase genes had originally been demonstrated to be induced after phosphate starvation of tomato cell culture but nothing was known about their expression or function in plants. We observed that the expression of the LX and LE genes is induced in leaves during an advanced stage of senescence with the LX transcript level being much more induced than that of LE. Low-level expression of the RNase genes was observed in flowers and artificially senescing detached leaves while no expression could be detected in stems, roots, or fruits at different ripening stages. Ethylene activated the LX gene expression in detached young leaves while LE gene expression, which could be transiently induced by wounding, appeared to be activated by abscisic acid. We suggest that the LX RNase has a role in RNA catabolism in the final stage of senescence, and LE may function during wounding as a plant defense protein.

Abscisic Acid↗
PubMed1998

Expressing foreign genes in the pistil: a comparison of S-RNase constructs in different Nicotiana backgrounds.

Transgenic plant experiments have great potential for extending our understanding of the role of specific genes in controlling pollination. Often, the intent of such experiments is to over-express a gene and test for effects on pollination. We have examined the efficiency of six different S-RNase constructs in Nicotiana species and hybrids. Each construct contained the coding region, intron, and downstream sequences from the Nicotiana alata S(A2)-RNase gene. Among the six expression constructs, two utilized the cauliflower mosaic virus (CaMV) 35S promoter with duplicated enhancer, and four utilized promoters from genes expressed primarily in pistils. The latter included promoters from the tomato Chi2;1 and 9612 genes, a promoter from the N. alata S(A2)-RNase gene, and a promoter from the Brassica SLG-13 gene. Some or all of the constructs were tested in N. tabacum, N. plumbaginifolia, N. plumbaginifolia x SI N. alata S(C10)S(c10) hybrids, N. langsdorffii, and N. langsdorffii x SC N. alata hybrids. Stylar specific RNase activities and S(A2)-RNase transcript levels were determined in transformed plants. Constructs including the tomato Chi2;1 gene promoter or the Brassica SLG-13 promoter provided the highest levels of S(A2)-RNase expression. Transgene expression patterns were tightly regulated, the highest level of expression was observed in post-anthesis styles. Expression levels of the S(A2)-RNase transgenes was dependent on the genetic background of the host. Higher levels of S(A2)-RNase expression were observed in N. plumbaginifolia x SC N. alata hybrids than in N. plumbaginifolia.

Cloning, Molecular↗
PubMed1999

Targeted RNases: a feasibility study for use in HIV gene therapy.

A targeted RNase would be ideal for gene therapy of several acquired and inherited disorders. Such an RNase may be engineered to contain a ribonucleolytic domain and a specific target RNA binding domain. To demonstrate the feasibility of this approach, an RNase targeted against human immunodeficiency virus (HIV) RNA--Tev-RNase T1--was designed and tested for its use in HIV-1 gene therapy. A human CD4+ T lymphoid (MT4) cell line and human peripheral blood lymphocytes (PBLs) were transduced with retroviral vectors lacking or expressing the tevT1 gene. Expression of enzymatically functional Tev-RNase T1 protein and its lack of toxicity was demonstrated in stable MT4 transductants. Compared with control cells lacking this protein, both transduced MT4 cells and PBLs expressing Tev-RNase T1 delayed HIV-1 replication. Tev-RNase T1 was shown to act after integration, since HIV-1 proviral DNA could be detected, but the amount of HIV-1 RNA produced in MT4 cells and PBLs was significantly decreased. This study demonstrates the feasibility of a targeted RNase strategy for therapeutic use.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Escherichia coli RNase III (rnc) autoregulation occurs independently of rnc gene translation.

Control of mRNA stability is an established means of regulating gene expression. However, the detailed mechanisms by which such control is achieved are only now emerging. In particular, there remains a question about the involvement of translation. Escherichia coli ribonuclease III (RNase III) negatively autoregulates expression of its own gene (rnc) approximately 10-fold, by cleaving the untranslated leader and initiating approximately 10-fold more rapid decay of the rnc mRNA, after which RNase III plays no further role. Here, we define the mechanism of this control further. Mutations that increase rnc gene translation abolish autoregulation by increasing the stability of the RNase III-cleaved transcript RNA approximately 10-fold, with no effect on the uncleaved species. Mutations that decrease translation destabilize the rnc mRNA in the presence or absence of RNase III. In so doing, they reveal a pathway of rnc transcript decay distinct from the RNase III-dependent pathway. Stability of a 'mini-rnc' transcript containing the rnc leader and only the first two codons of the rnc gene is unaffected by decreased translation, presumably because sequences required for this pathway were removed. Importantly, this mini-rnc transcript is regulated normally by RNase III. Moreover, rnc transcripts synthesized in vitro do not decay in cell-free extracts lacking ribosomes, unless they are first cleaved by RNase III. These two results show that RNase III cleavage can initiate rnc transcript decay independently of rnc gene translation, unambiguously establishing that control of mRNA decay need not involve changes in translation. How rnc gene translation is optimized for efficient autoregulation will also be discussed.

Base Sequence↗
PubMed2001

Autoregulation allows Escherichia coli RNase E to adjust continuously its synthesis to that of its substrates.

The Escherichia coli endonuclease RNase E plays a key role in rRNA maturation and mRNA decay. In particular, it controls the decay of its own mRNA by cleaving it within the 5'-untranslated region (UTR), thereby autoregulating its synthesis. Here, we report that, when the synthesis of an RNase E substrate is artificially induced to high levels in vivo, both the rne mRNA concentration and RNase E synthesis increase abruptly and then decrease to a steady-state level that remains higher than in the absence of induction. Using rne-lacZ fusions that retain or lack the rne 5'UTR, we show that these variations reflect a transient mRNA stabilization mediated by the rne 5'UTR. Finally, by putting RNase E synthesis under the control of an IPTG-controlled promoter, we show that a similar, rne 5'UTR-mediated mRNA stabilization can result from a shortage of RNase E. We conclude that the burst in substrate synthesis has titrated RNase E, stabilizing the rne mRNA by protecting its 5'UTR. However, this stabilization is self-correcting, because it allows the RNase E pool to expand until its mRNA is destabilized again. Thus, autoregulation allows RNase E to adjust its synthesis to that of its substrates, a behaviour that may be common among autoregulated proteins. Incidentally, this adjustment cannot occur when translation is blocked, and we argue that the global mRNA stabilization observed under these conditions originates in part from this defect.

5' Untranslated Regions↗
PubMed2002

Consequences of RNase E scarcity in Escherichia coli.

The endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate-determining. Our data also indicate that E. coli growth requires a cellular RNase E concentration at least 10-20% of normal and that the feedback mechanism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the in-crease in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, when either mutated or cloned on a multicopy plasmid, seems to be capable of compensating for an inadequate supply of this essential protein.

Cell Division↗
PubMed1998

A relic S-RNase is expressed in the styles of self-compatible Nicotiana sylvestris.

We surveyed ribonuclease activity in the styles of Nicotiana spp. and found little or no activity in self-compatible species and in a self-compatible accession of a self-incompatible species. All self-incompatible species had high levels of ribonuclease activity in their style. Interestingly, one self-compatible species, N. sylvestris, had a level of stylar ribonuclease activity comparable to that of some self-incompatible Nicotiana species. A ribonuclease with biochemical properties similar to those of the self-incompatibility (S-)RNases of N. alata was purified from N. sylvestris styles. The N-terminal sequence of this protein was used to confirm the identity of a cDNA corresponding to the stylar RNase. The amino acid sequence deduced from the cDNA was related to those of the S-RNases and included the five conserved regions characteristic of these proteins. It appears that the N. sylvestris RNase may have evolved from the S-RNases and is an example of a 'relic S-RNase'. A number of features distinguish the N. sylvestris RNase from the S-RNases, and the role these may have played in the presumed loss of the self-incompatibility response during the evolution of this species are discussed.

Amidohydrolases↗
PubMed2005

RNase P cleaves transient structures in some riboswitches.

RNase P from Escherichia coli cleaves the coenzyme B12 riboswitch from E. coli and a similar one from Bacillus subtilis. The cleavage sites do not occur in any recognizable structure, as judged from theoretical schemes that have been drawn for these 5' UTRs. However, it is possible to draw a scheme that is a good representation of the E. coli cleavage site for RNase P and for the cleavage site in B. subtilis. These data indicate that transient structures are important in RNase P cleavage and in riboswitch function. Coenzyme B12 has a small inhibitory effect on E. coli RNase P cleavage of the E. coli riboswitch. Both E. coli RNase P and a partially purified RNase P from Aspergillus nidulans mycelia succeeded in cleaving a putative arginine riboswitch from A. nidulans. The cleavage site may be a representative of another model substrate for eukaryotic RNase P. This 5' UTR controls splicing of the arginase mRNA in A. nidulans. Four other riboswitches in E. coli were not cleaved by RNase P under the conditions tested.

Aspergillus nidulans↗
PubMed2006

Functional reconstitution and characterization of Pyrococcus furiosus RNase P.

RNase P, which catalyzes the magnesium-dependent 5'-end maturation of tRNAs in all three domains of life, is composed of one essential RNA and a varying number of protein subunits depending on the source: at least one in bacteria, four in archaea, and nine in eukarya. To address why multiple protein subunits are needed for archaeal/eukaryal RNase P catalysis, in contrast to their bacterial relative, in vitro reconstitution of these holoenzymes is a prerequisite. Using recombinant subunits, we have reconstituted in vitro the RNase P holoenzyme from the thermophilic archaeon Pyrococcus furiosus (Pfu) and furthered our understanding regarding its functional organization and assembly pathway(s). Whereas Pfu RNase P RNA (RPR) alone is capable of multiple turnover, addition of all four RNase P protein (Rpp) subunits to Pfu RPR results in a 25-fold increase in its k(cat) and a 170-fold decrease in K(m). In fact, even in the presence of only one of two specific pairs of Rpps, the RPR displays activity at lower substrate and magnesium concentrations. Moreover, a pared-down, mini-Pfu RNase P was identified with an RPR deletion mutant. Results from our kinetic and footprinting studies on Pfu RNase P, together with insights from recent structures of bacterial RPRs, provide a framework for appreciating the role of multiple Rpps in archaeal RNase P.

Base Sequence↗
PubMed2002

Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts.

Misincorporated ribonucleotides in DNA will cause DNA backbone distortion and may be targeted by DNA repair enzymes. Using double-stranded oligonucleotide probes containing a single ribose, we demonstrate a robust activity in human, yeast, and Escherichia coli cell-free extracts that nicks 5' of the ribose. The human and yeast extracts also make a subsequent cut 3' of the ribonucleotide releasing a ribonucleotide monophosphate. The resulting 1-nt gap is an ideal substrate for polymerase and ligase to complete a proposed repair sequence that effectively replaces the ribose with deoxyribose. Screening of yeast deletion mutant cells reveals that the initial nick is made by RNase H(35), a RNase H type 2 enzyme, and the second cut is made by Rad27p, the yeast homologue of human FEN-1 protein. RNase H type 2 enzymes are present in all kingdoms of life and are evolutionarily well conserved. We knocked out the corresponding rnhb gene in E. coli and show that extracts from this strain lack the nicking activity. Conversely, a highly purified archaeal RNase HII type 2 protein has a pronounced activity. To study substrate specificity, extracts were made from a yeast double mutant lacking the other main RNase H enzymes [RNase H1 and RNase H(70)], while maintaining RNase H(35). It was found that a single ribose is preferred as substrate over a stretch of riboses, further strengthening a proposed role of this enzyme in the repair of misincorporated ribonucleotides rather than (or in addition to) processing RNADNA hybrid molecules.

Cell-Free System↗
PubMed1985

RNase T is responsible for the end-turnover of tRNA in Escherichia coli.

A mutant strain deficient in RNase T was isolated and used to study the role of this enzyme in Escherichia coli. Strains lacking as much as 70% of RNase T activity, alone or in combination with the absence of other RNases, display normal growth properties. However, in cca strains, which lack tRNA nucleotidyltransferase, RNase T-deficient derivatives accumulate lower levels of defective tRNA and grow at increased rates compared to their RNase T+ parents. Slow-growing cca strains revert to a faster-growing form that contains less defective tRNA but which is still cca. All of these strains have decreased levels of RNase T. These data indicate that RNase T is responsible for nucleotide removal during the tRNA end-turnover process and that the amount of defective tRNA in cells is determined by the relative levels of RNase T and tRNA nucleotidyltransferase.

Bacterial Proteins↗