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PubMed2000

Architecture and function of the human endonucleases RNase P and RNase MRP.

In the past decade, important advances have been made in our knowledge of the composition of human RNase MRP and RNase P complexes. Both ribonucleoprotein particles function as endonucleases and contain RNA components that are structurally related. RNase MRP has been suggested to be involved in the processing of precursor rRNA; RNase P, in the maturation of tRNA. Here we give an overview of current data on the structure and function of human RNase MRP and RNase P particles, with emphasis on their molecular composition. At present, seven protein subunits, probably all associated with both ribonucleoprotein particles, have been isolated and their corresponding cDNAs cloned. Although no known structural motifs can be identified in the amino acid sequences of these proteins, the majority is clearly rich in basic residues. For two protein subunits, a cluster of basic amino acids have been shown to be involved in nucleolar accumulation, whereas another protein, which lacks such a region, probably enters the nucleolus by way of a piggyback mechanism. The binding regions for several of the protein subunits on the RNA have been identified, and the data have been used to create a putative structural model for the RNase MRP particle. The rather obscure situation concerning the association of the autoantigenic Th-40 protein and its possible relationship with one of the subunits, Rpp38, is discussed.

Autoantigens↗

PubMed1997

Tissue-specific expression of pancreatic-type RNases and RNase inhibitor in humans.

The tissue-specific expression of five human pancreatic-type RNases and RNase inhibitor was analyzed by Northern hybridization against poly(A)+ RNA prepared from 16 normal tissues. The widespread expression of RNase 1 was observed in almost all of the tissues. RNase 4 and angiogenin showed a similar distribution of expression abundantly present in the liver. This suggested the identity of the cell types producing these two molecules. However, no relativity appeared to be present between the vascularization of the tissues and the angiogenin expression. A narrow range of expression of the eosinophil-derived neurotoxin gene was observed. This localization seems related to the phagocytic cells in the tissues. The undetectable level of the eosinophil cationic protein mRNA in normal tissues suggests that the differentiation of eosinophils, triggered by inflammation and/or atopy, is required. The expression of RNase inhibitor was found to be ubiquitous. The regulatory function of inhibitor against RNases in the cell should be considered in studying the physiological significance of the pancreatic-type RNase family.

DNA, Complementary↗

PubMed1991

Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme thermophile Thermus thermophilus HB8: a thermostable RNase H can functionally replace the Escherichia coli enzyme in vivo.

A DNA fragment encoding Ribonuclease H (EC 3. 1.26.4) was isolated from an extreme thermophilic bacterium, Thermus thermophilus HB8, by its ability to complement the temperature-sensitive growth of an Escherichia coli rnhA deficient mutant. The primary amino acid sequence showed 56% similarity to that of E. coli RNase HI but little or no homology to E. coli RNase HII. Enzymes derived from thermophilic organisms tend to have fewer cysteines than their bacterial counterparts. However, T. thermophilus RNase H has one more cysteine than its E. coli homologue. Stability of the RNase H in extracts of T. thermophilus to elevated temperatures was the same for the protein expressed in E. coli. T. thermophilus RNase H should, therefore, be a useful tool for editing RNA-DNA hybrid molecules at higher temperatures and may also be stable enough to be used in a cyclical process. It was suggested that regulation of expression of the RNase H may be different from that of E. coli. RNase HI.

Amino Acid Sequence↗

PubMed2001

Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B. subtilis RNase P protein.

The bacterial RNase P holoenzyme catalyzes the formation of the mature 5'-end of tRNAs and is composed of an RNA and a protein subunit. Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contains the active site of this ribozyme. We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examined the catalytic activity of this C-domain-P protein complex. The C-domain forms a specific complex with the P protein with a binding constant of approximately 0.1 microM. The C-domain-P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (approximately 0.9 min(-1) at pH 7.8) and substrates with a hairpin-loop 3' to the cleavage site (approximately 40 min(-1)). The holoenzyme reaction is much more efficient with a pre-tRNA substrate, binding at least 100-fold better and cleaving 10-500 times more efficiently. These results demonstrate that the RNase P holoenzyme is functionally constructed in three parts. The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates. The specificity and affinity for the substrate is generated by either the specificity domain of RNase P RNA binding to a T stem-loop-like hairpin or RNase P protein binding to a single-stranded RNA. This modular construction may be exploited to obtain RNase P-based ribonucleoprotein complexes with altered substrate specificity.

Bacillus subtilis↗

PubMed1994

pH profile of kinetic constants of RNase Rh from Rhizopus niveus and its mutant enzymes towards UpU, and possible mechanisms of RNase Rh.

In order to elucidate the mechanism of action of Rhizopus niveus RNase Rh, we investigated the pH profiles of the kinetic parameters of RNase RNAP Rh, a derivative of RNase Rh, and its mutant enzymes, i.e., RNase RNAP Rh H104F, RNase RNAP Rh E105Q, and RNase RNAP Rh D51N. Based on comparisons of their profiles we concluded that protonation of His104 is indispensable for the enzymatic activity and Glu105 accelerates the enzymatic activity, especially at acid pH centered at pH 3.5. Based on these data and the previous data on the chemical modification and enzymatic properties of other mutant enzymes, we propose the following as a possible mechanisms of RNase Rh action. (i) His109 participates in enzymatic action as a general base catalyst which removes the hydrogen of the 2'-OH of the ribose moiety. (ii) His46 participates in the reaction as a general acid catalyst which interacts with the 5'-oxygen atom of the scissile phosphodiester bond and becomes a proton donor to the departing nucleoside or nucleotide. (iii) His104 interacts with phosphate anion and its protonation is favorable for the enzymatic activity. (iv) Since the protonated form of Glu105 is more favorable for activity, we postulate two possible roles for Glu105: (a) its stabilizes the intermediate, and (b) it interacts with the oxygen atom of P = O and polarizes the phosphorus atom.

Dinucleoside Phosphates↗

PubMed1997

A Mutant S3 RNase of Petunia inflata Lacking RNase Activity Has an Allele-Specific Dominant Negative Effect on Self-Incompatibility Interactions.

Gametophytic self-incompatibility in the Solanaceae is controlled by a multiallelic locus called the S locus. Growth of pollen tubes in the pistil is inhibited when the pollen has one of the two S alleles carried by the pistil. The products of a number of pistil S alleles[mdash]S proteins or S RNases[mdash]have been identified, and their role in controlling the pistil's ability to reject self-pollen has been positively established. In contrast, the existence of pollen S allele products has so far been inferred entirely from genetic evidence. Here, we introduced a modified S3 gene of Petunia inflata encoding an S3 RNase lacking RNase activity into P. inflata plants of the S2S3 genotype to determine whether the production of the mutant protein, designated S3(H93R), would have any effect on the ability of the transgenic plants to reject S2 and S3 pollen. Analysis of the self-incompatibility behavior of 49 primary transgenic plants and the progeny of three plants (H30, H37, and H40) that produced S3(H93R) in addition to producing wild-type levels of endogenous S2 and S3 RNases revealed that S3(H93R) had a dominant negative effect on the function of the S3 RNase in rejecting self-pollen; however, it had no effect on the function of the S2 RNase. One likely explanation of the results is that S3(H93R) competes with the S3 RNase for binding to a common molecule, which is presumably the product of the pollen S3 allele.

Journal Article↗

PubMed1991

A gene required for RNase P activity in Candida (Torulopsis) glabrata mitochondria codes for a 227-nucleotide RNA with homology to bacterial RNase P RNA.

We have mapped a gene in the mitochondrial DNA of Candida (Torulopsis) glabrata and shown that it is required for 5' end maturation of mitochondrial tRNAs. It is located between the tRNAfMet and tRNAPro genes, the same tRNA genes that flank the mitochondrial RNase P RNA gene in the yeast Saccharomyces cerevisiae. The gene is extremely AT rich and codes for AU-rich RNAs that display some sequence homology with the mitochondrial RNase P RNA from S. cerevisiae, including two regions of striking sequence homology between the mitochondrial RNAs and the bacterial RNase P RNAs. RNase P activity that is sensitive to micrococcal nuclease has been detected in mitochondrial extracts of C. glabrata. An RNA of 227 nucleotides that is one of the RNAs encoded by the gene that we mapped cofractionated with this mitochondrial RNase P activity on glycerol gradients. The nuclease sensitivity of the activity, the cofractionation of the RNA with activity, and the homology of the RNA with known RNase P RNAs lead us to propose that the 227-nucleotide RNA is the RNA subunit of the C. glabrata mitochondrial RNase P enzyme.

Base Sequence↗

PubMed1996

hPop1: an autoantigenic protein subunit shared by the human RNase P and RNase MRP ribonucleoproteins.

The eukaryotic endonucleases RNase P and RNase MRP require both RNA and protein subunits for function. Even though the human RNase P and MRP RNAs were previously characterized, the protein composition of the particles remains unknown. We have identified a human a Caenorhabditis elegans sequence showing homology to yPop1, a protein subunit of the yeast RNase P and MRP particles. A cDNA containing the complete coding sequence for the human protein, hPop1, was cloned. Sequence analysis identifies three novel sequence motifs, conserved between the human, C. elegans and yeast proteins. Affinity-purified anti-hPop1 antibodies recognize a single 115 kDa protein in HeLa cell nuclear extracts. Immunoprecipitations with different anti-hPop1 antibodies demonstrate an association of hPop1 with the vast majority of the RNase P and MRP RNAs in HeLa cell nuclear extracts. Additionally, anti-hPop1 immunoprecipitates possess RNase P enzymatic activity. These results establish hPop1 as the first identified RNase P and MRP protein subunit from humans. Anti-hPop1 antibodies generate a strong nucleolar and a weaker homogeneous nuclear staining in HeLa cells. A certain class of autoimmune patient serum precipitates in vitro-translated hPop1. hPop1 is therefore an autoantigen in patients suffering from connective tissue diseases.

Amino Acid Sequence↗

PubMed1993

Definition of the Th/To ribonucleoprotein by RNase P and RNase MRP.

We show that the Th/To ribonucleoprotein is defined by (i) the co-immunoprecipitation of two RNAs, (ii) the co-immunoprecipitation of four major polypeptides and (iii) the quantitative immune recognition of both RNase P and RNase MRP. No serum was found that recognizes either one of these two enzymes exclusively. The specific co-immunoprecipitation of RNase MRP and RNase P by all Th/To ribonucleoprotein autoantibodies indicates that the anti-Th/To autoimmune response is directed against both enzymes in a quantitatively indistinguishable manner. Thus the Th/To ribonucleoprotein is defined by RNase P and RNase MRP.

Antibody Specificity↗

PubMed2000

The 3' substrate determinants for the catalytic efficiency of the Bacillus subtilis RNase P holoenzyme suggest autolytic processing of the RNase P RNA in vivo.

We investigated the catalytic efficiency and the specificity of the Bacillus subtilis RNase P holoenzyme reaction with substrates that contain a single strand, a hairpin loop, or a tRNA 3' to the cleavage site. At a saturating ribozyme concentration, RNase P can cleave a single-stranded RNA at approximately 0.6 min(-1) at pH 7.8. Replacing the single-stranded RNA 3' to the cleavage site by a hairpin loop or by the yeast tRNA(Phe) increases the cleavage rate by up to approximately 600-fold and approximately 3,200-fold, respectively. These results show that compared to a single-stranded RNA substrate, the cleavage rate for the holoenzyme reaction is primarily enhanced by an acceptor-stem-like helix. Substrate binding, approximately 7-10 microM for a single-stranded RNA, improves by approximately 1,000-fold upon the addition of the tRNA. The efficiency of the RNase P holoenzyme cleaving a single-stranded RNA is sufficiently high to consider autolytic processing of the RNase P RNA (denoted P RNA) transcript in the cell. The addition of the RNase P protein to a precursor form of the P RNA in vitro results in autolytic processing of the 5' and the 3' end of this precursor in a matter of minutes. Autolytic processing produces the reported 5' end of the mature P RNA. The precise 3' end generated by autolytic processing is different over the course of the reaction and the final product is 4 nt shorter than the reported 3' end of the B. subtilis P RNA. The observed 3' end in vitro is consistent with the property of the holoenzyme reaction with single-stranded RNA substrates. The discrepancy with the reported 3' end may be due to other processing events in vivo or inaccurate determination of the mature 3' end of the P RNA isolated from the cell. We propose that the mature B. subtilis P RNA is generated at least in part by autolytic processing upon the binding of the RNase P protein to the precursor P RNA.

Bacillus subtilis↗

PubMed1997

The RNase P RNA from cyanobacteria: short tandemly repeated repetitive (STRR) sequences are present within the RNase P RNA gene in heterocyst-forming cyanobacteria.

The RNase P RNA gene (rnpB) from 10 cyanobacteria has been characterized. These new RNAs, together with the previously available ones, provide a comprehensive data set of RNase P RNA from diverse cyanobacterial lineages. All heterocystous cyanobacteria, but none of the non-heterocystous strains analyzed, contain short tandemly repeated repetitive (STRR) sequences that increase the length of helix P12. Site-directed mutagenesis experiments indicate that the STRR sequences are not required for catalytic activity in vitro. STRR sequences seem to have recently and independently invaded the RNase P RNA genes in heterocyst-forming cyanobacteria because closely related strains contain unrelated STRR sequences. Most cyanobacteria RNase P RNAs lack the sequence GGU in the loop connecting helices P15 and P16 that has been established to interact with the 3'-end CCA in precursor tRNA substrates in other bacteria. This character is shared with plastid RNase P RNA. Helix P6 is longer than usual in most cyanobacteria as well as in plastid RNase P RNA.

Base Composition↗

PubMed2000

Eukaryotic ribonucleases HI and HII generate characteristic hydrolytic patterns on DNA-RNA hybrids: further evidence that mitochondrial RNase H is an RNase HII.

RNase H activities from HeLa cells (either of cytoplasmic or mitochondrial origin), and from mitochondria of beef heart and Xenopus ovaries, have been tested with RNA-DNA substrates of defined length (20 bp) and sequence. Substrates were either blunt-ended, or presented DNA or RNA overhangs. The hydrolysis profiles obtained at early times of the digestion showed a good correlation between the class of RNase H, either type I or II assigned according to biochemical parameters, whatever the organism. Consequently, the pattern of primary cuts can be considered as a signature of the predominant RNase H activity. For a given sequence, hydrolysis profiles obtained are similar, if not identical, for either blunt-ended substrates or those presenting overhangs. However, profiles showed variations depending on the sequence used. Of the three sequences tested, one appears very discriminatory, class I RNases H generating a unique primary cut 3 nt from the 3' end of the RNA strand, whereas class II RNases H generated two simultaneous primary cuts at 6 and at 8 nt from the 5' end of the RNA strand. Hydrolysis profiles further confirm the assignation of the mitochondrial RNase H activity from HeLa cells, beef heart and Xenopus oocytes to the class II.

Animals↗

PubMed1995

RNase E autoregulates its synthesis by controlling the degradation rate of its own mRNA in Escherichia coli: unusual sensitivity of the rne transcript to RNase E activity.

RNase E is a key regulatory enzyme that appears to control the principal pathway for mRNA degradation in Escherichia coli. Here, we show that RNase E represses its own synthesis by reducing the cellular concentration of the rne (RNase E) gene transcript. Autoregulation is achieved by modulating the longevity of this 3.6-kb mRNA, whose half-life ranges from < 40 sec to > 8 min depending on the level of RNase E activity in the cell. Feedback regulation is mediated in cis by the 5'-terminal 0.44-kb segment of rne mRNA, which is sufficient to confer this property onto a heterologous transcript to which it is fused. Like the intact protein, an amino-terminal fragment of RNase E lacking 563 amino acid residues can act in trans to repress rne gene expression. Paradoxically, raising the rne gene copy number 21-fold in E. coli causes an unexpected reduction in the concentration of the full-length rne transcript, yet results in a small increase in RNase E protein production. These surprising phenomena are explained in terms of a model in which the degradation of this long and highly labile mRNA commences before elongation of the nascent transcript has been completed. In such circumstances, gene expression can be unusually sensitive to changes in mRNA stability.

Base Sequence↗

PubMed1988

15N- and 1H-NMR investigations of the active-site amino acids in semisynthetic RNase S' and RNase A.

Extensive 15N-NMR investigations of active-site amino acids were made possible by the solid-phase synthesis of the N-terminal pentadecapeptide of RNase A with selectively 15N-enriched amino acids. On complexation with S-protein a fully active RNase S' complex was obtained. The 15N resonances of the side chains of lysine-7 (N epsilon), glutamine-11 (N gamma), and histidine-12 (N pi, tau) were studied in the free synthetic peptide, in the RNase S' complex and in the nucleotide complexes RNase S' with 2'CMP, 3'CMP, and 5'AMP. The analysis of the 15N-1H couplings, the 15N line broadenings due to proton exchange, and the chemical shift values showed that, while the imidazole ring is directly involved in the peptide-protein interaction, the side chains of Lys-7 and Gln-11 do not contribute to this interaction. In the nucleotide complexes the resonances of His-12 and Gln-11 are shifted downfield. In the 2'CMP complex a doublet for the N tau signal of His-12 indicates a stable H bond between this nitrogen and the phosphate group of nucleotide. The other nucleotide influence the resonances of the imidazole group much less, possibly due to a slightly different orientation of the phosphate group. The downfield shift of the Gln-11 resonance indicates an interaction between the carbonyl oxygen of the amide group and the phosphate moiety of the nucleotide. The only observable effect of nucleotide complexation on the Lys-7 signal is line broadening due to reduced proton exchange. For comparison with the 15N-NMR titration curves of His-12 in RNase S' the 1H-NMR titration curves of RNase A were also recorded. Both shape and pK values were very similar for the 15N and the 1H titration curves. An extensive analysis of the protonation equilibria with several fitting models showed that a mutual interaction of the imidazole groups of the active-site histidines results in flat titration curves. The Hill plots of all resonances of the imidazole rings, including the 15N resonances, show a small inflection in the pH range 5.8-6.4. Since the existence of a diimidazole system is most likely in this pH range, the inflection could be interpreted as a disturbance of the mutual electrostatic interaction of the active-site histidines by a partial H-bond formation between the imidazole groups.

Amino Acids↗

PubMed1989

The RNA processing enzyme RNase MRP is identical to the Th RNP and related to RNase P.

Sera from patients with autoimmune diseases often contain antibodies that bind ribonucleoproteins (RNPs). Sera from 30 such patients were found to immunoprecipitate ribonuclease P (RNase P), an RNP enzyme required to process the 5' termini of transfer RNA transcripts in nuclei and mitochondria of eukaryotic cells. All 30 sera also immunoprecipitated the nucleolar Th RNP, indicating that the two RNPs are structurally related. Nucleotide sequence analysis of the Th RNP revealed it was identical to the RNA component of the mitochondrial RNA processing enzyme known as RNase MRP. Antibodies that immunoprecipitated the Th RNP selectively depleted murine and human cell extracts of RNase MRP activity, indicating that the Th and RNase MRP RNPs are identical. Since RNase P and RNase MRP are not associated with each other during biochemical purification, we suggest that these two RNA processing enzymes share a common autoantigenic polypeptide.

Autoantigens↗

PubMed2006

Investigation of conformational changes induced by binding of pancreatic RNase to anti-RNase IgG derived Fab monomer using optical procedures.

The conformational changes induced in Fab fragments of polyclonal anti-RNase antibody molecules obtained by digestion with papain as a result of binding of pancreatic RNase have been studied. The RNase-Fab complex (RN-Fab), being soluble, could be subjected to thermodynamic investigations using optical strategies, also because of the absence of tryptophan in RNase. Internalization of the chromophores (tryptophans and tyrosines) of Fab occurs when it binds to RNase, suggesting an increase in the compactness of Fab due to the binding of RNase.

Animals↗

PubMed1977

Action of A RNase and AS RNase on growth of cells in vitro.

The effect of bovine pancreatic A RNase and bull seminal vesicle AS RNase on proliferation of HeLa cells and LEP cells in vitro was studied. The ribonucleases were used in doses of 0.1, 1.0, 10.0, and 100.0 microgram per ml of medium and 100 000 inoculated cells. Proliferation was evaluated by the growth curves. Both in single and long-term experiments. A RNase in all doses used had no effect on cell proliferation, whereas AS RNase exerted inhibitory activity beginning with the dose of 10 microgram, and affected more markedly the LEP cells (a diploid non-malignant cell line) than the malignant heteroploid HeLa cells. When added to the cell suspension, AS RNase was shown by indirect immunofluorescence to bind to HeLa cell membranes in all phases of growth, whereas AS RNase was found on membranes of LEP cells only when they were derived from the log phase of growth.

Animals↗

PubMed2001

The basic loop of the RNase H domain of MLV RT is important both for RNase H and for polymerase activity.

Escherichia coli RNase H has a basic extension that is involved in binding nucleic acid substrates. This basic extension is present in the RNase H of Moloney murine leukemia virus reverse transcriptase (MLV RT), but has been deleted from the RNase H of HIV-1 RT. Previous work showed that removing the basic loop from MLV RT (the mutant is called DeltaC) blocked viral replication; however, DeltaC MLV RT retained RNase H activity in an in situ gel assay. We prepared recombinant DeltaC MLV RT and compared its activity to wild-type MLV RT. The DeltaC mutant is impaired in both polymerase and RNase H activity; the pattern of defects suggests that the basic loop is involved in the binding of MLV RT to a heteropolymeric template-primer.

Animals↗