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At least 307 records · Page 17Linked to original sources
PubMed1993

Expression of gene 19 of the conjugative plasmid R1 is controlled by RNase III.

Specific cleavage of mRNAs by RNase III has been shown to control the expression of several Escherichia coli genes. We show here that the expression of gene 19 of the conjugative resistance plasmid R1 is controlled in its expression by the same endoribonuclease. In vivo studies revealed that a DNA fragment of 150 nucleotides including a perfect 22 nucleotide inverted repeat in the gene 19 coding region is responsible for the low expression of the gene both at the protein and the RNA levels. By using a translational gene 19-lacZ fusion in isogenic RNase III+ and RNase III- strains we could identify RNase III as the key element in the down-regulation of gene 19 expression. The sequencing of in vitro generated and RNase III-digested transcripts confirmed the in vivo studies and revealed the exact positions of the RNase III cleavage sites within the coding part of the gene 19 transcript. The in vitro determined RNase III cleavage of gene 19 mRNA was confirmed by in vivo primer extension analysis. Finally, we could show that an exchange of three nucleotides within the RNase III recognition site abolished RNase III cleavage in vitro.

Amino Acid Sequence↗
PubMed2005

Stylar glycoproteins bind to S-RNase in vitro.

S-RNases determine the specificity of S-specific pollen rejection in self-incompatible plants of the Solanaceae, Rosaceae, and Scrophulariaceae. They are also implicated in at least two distinct types of unilateral interspecific incompatibility in Nicotiana. However, S-RNase itself is not sufficient for most types of pollen rejection, and evidence for its direct interaction with pollen tubes is limited. Thus, non-S-RNase factors also are required for pollen rejection. As one approach to identifying such factors, we tested whether SC10-RNase from Nicotiana alata would bind to other stylar proteins in vitro. SC10-RNase was immobilized on Affi-gel, and binding proteins were analyzed by SDS-PAGE and immunoblotting. In addition to SC10-RNase and a small protein similar to lily chemocyanin, the most prominent binding proteins include NaTTS, 120K, and NaPELPIII, these latter three being arabinogalactan proteins previously shown to interact directly with pollen tubes. We also show that SC10-RNase and these glycoproteins migrate as a complex in a native PAGE system. Our hypothesis is that S-RNase forms a complex with these glycoproteins in the stylar ECM, that the glycoproteins interact directly with the pollen tubes and thus that the initial interaction between the pollen tube and S-RNase is indirect.

Amino Acid Sequence↗
PubMed1987

Processing and secretion of the Yarrowia lipolytica RNase.

Secretion of the extracellular RNase from the yeast Yarrowia lipolytica was studied in pulse-chase and immunoprecipitation experiments. A polypeptide of 45,000 daltons was immunoprecipitated from [35S]methionine-labeled cell extracts and supernatant medium by rabbit anti-RNase antiserum. The RNase was secreted rapidly; the time between synthesis and appearance in the extracellular medium was about 5 min. In pulse-chase experiments, about 50% of the RNase was still cell associated 30 min after labeling. A polypeptide of 73,000 daltons whose immunoprecipitation was blocked by an excess of purified RNase was also detected. It broke down to a polypeptide with the same mobility and same peptide map as the mature RNase. Peptide maps of the undegraded 73-kilodalton polypeptide and the intracellular mature RNase contained several peptides of identical mobility. Immunoprecipitates from cells labeled in the presence of tunicamycin contained 66- and 45-kilodalton polypeptides. Endoglycosidase H treatment of the 73-kilodalton polypeptide converted it to a 66-kilodalton form, but did not change the apparent molecular weight of the mature form of the RNase. Labeling kinetics from pulse-chase experiments did not clearly support a precursor-product relationship between the 73-kilodalton polypeptide and the intracellular 45-kilodalton form of the RNase, and other relationships between the two polypeptides are possible.

Carbohydrates↗
PubMed1988

Cloning, characterization, and effects of overexpression of the Escherichia coli rnd gene encoding RNase D.

RNase D is a 3'-exoribonuclease whose in vitro specificity has suggested that it is involved in the processing of tRNA precursors. Its in vivo role has remained unclear, however, because mutant cells devoid of the enzyme display no defect in growth or tRNA processing. To learn more about the structure and function of RNase D, we cloned the Escherichia coli rnd gene, which is thought to code for this enzyme. The rnd gene was isolated from a cosmid library based on elevated RNase D activity and was subcloned as a 1.4-kilobase-pair fragment in pUC18. Maxicell analysis of the cloned fragment revealed that a single protein of approximately 40 kilodaltons, which is the size of RNase D, was synthesized. The rnd gene is present as a single copy on the E. coli chromosome and is totally absent in a deletion mutant. Cells that harbored the cloned rnd gene displayed RNase D activity that was elevated as much as 20-fold over that of the wild type. As growth of the culture progressed, however, RNase D specific activity declined dramatically, together with a similar decrease in plasmid copy number. In contrast, no decrease in copy number was observed with an inactive rnd gene. Placement of the rnd gene downstream from the lac promoter led to inducible RNase D overexpression and concomitantly slowed cell growth. These findings support the idea that rnd is the structural gene for RNase D and indicate that elevated RNase D activity is deleterious to E. coli.

Cloning, Molecular↗
PubMed1988

Expression of double-stranded-RNA-specific RNase III of Escherichia coli is lethal to Saccharomyces cerevisiae.

The gene for the double-stranded RNA (dsRNA)-specific RNase III of Escherichia coli was expressed in Saccharomyces cerevisiae to examine the effects of this RNase activity on the yeast. Induction of the RNase III gene was found to cause abnormal cell morphology and cell death. Whereas double-stranded killer RNA is degraded by RNase III in vitro, killer RNA, rRNA, and some mRNAs were found to be stable in vivo after induction of RNase III. Variants selected for resistance to RNase III induction were isolated at a frequency of 4 X 10(-5) to 5 X 10(-5). Ten percent of these resistant strains had concomitantly lost the capacity to produce killer toxin and M dsRNA while retaining L dsRNA. The genetic alteration leading to RNase resistance was localized within the RNase III-coding region but not in the yeast chromosome. These results indicate that S. cerevisiae contains some essential RNA which is susceptible to E. coli RNase III.

Cloning, Molecular↗
PubMed2000

Regions of RNase E important for 5'-end-dependent RNA cleavage and autoregulated synthesis.

RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in Escherichia coli. Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5' end. Here we show that the preference of E. coli RNase E for 5'-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related E. coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from Synechocystis, indicating that the 5'-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5'-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E. coli. Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E. coli cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels.

Amino Acid Sequence↗
PubMed1998

Inactivation of the RNase activity of glycoprotein E(rns) of classical swine fever virus results in a cytopathogenic virus.

Envelope glycoprotein E(rns) of classical swine fever virus (CSFV) has been shown to contain RNase activity and is involved in virus infection. Two short regions of amino acids in the sequence of E(rns) are responsible for RNase activity. In both regions, histidine residues appear to be essential for catalysis. They were replaced by lysine residues to inactivate the RNase activity. The mutated sequence of E(rns) was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact E(rns), the mutated proteins had lost their RNase activity. The mutated proteins reacted with E(rns)-specific neutralizing monoclonal and polyclonal antibodies and were still able to inhibit infection of swine kidney cells (SK6) with CSFV, but at a concentration higher than that measured for intact E(rns). This result indicated that the conformation of the mutated proteins was not severely affected by the inactivation. To study the effect of these mutations on virus infection and replication, a CSFV mutant with an inactivated E(rns) (FLc13) was generated with an infectious DNA copy of CSFV strain C. The mutant virus showed the same growth kinetics as the parent virus in cell culture. However, in contrast to the parent virus, the RNase-negative virus induced a cytopathic effect in swine kidney cells. This effect could be neutralized by rescue of the inactivated E(rns) gene and by neutralizing polyclonal antibodies directed against E(rns), indicating that this effect was an inherent property of the RNase-negative virus. Analyses of cellular DNA of swine kidney cells showed that the RNase-negative CSFV induced apoptosis. We conclude that the RNase activity of envelope protein E(rns) plays an important role in the replication of pestiviruses and speculate that this RNase activity might be responsible for the persistence of these viruses in their natural host.

Animals↗
PubMed1990

Comparative pyrimidine- and purine-specific RNAse-gold labeling on pancreatic acinar cells and isolated hepatocytes.

We applied the enzyme-gold approach to investigate the potential of various ribonucleases displaying different affinities for ultrastructural localization of particular RNA molecules. Five specific ribonucleases were used: three from a pancreatic source, RNAses A, B, and S with affinities for pyrimidine bases; and two from Aspergillus oryzae, RNAses T1 and T2 specific for purine bases. Conditions required for preparing each RNAse-gold complex, as well as for obtaining specific labelings, were determined. Application of the probes on thin sections of pancreatic acinar cells yielded labeling patterns that differed according to the enzyme used. Pancreatic RNAses labeled mostly the rough endoplasmic reticulum and the nucleolus, whereas fungal RNAses labeled more intensely the interchromatin space and the nucleolus, the rough endoplasmic reticulum being labeled to a lesser extent. Areas rich in interchromatin granules were intensely labeled by the RNAses T1 and T2. This was confirmed on DRB-treated hepatocytes, which displayed large clusters of interchromatin granules. Perichromatin granules were labeled by the RNAse A- and T1-gold complexes. These results provide a strong indication for the presence of RNA molecules in both types of granules. Nuclear pores were labeled, particularly by the RNAses T1 and T2, thus supporting the hypothesis for the site of RNA transit between nucleus and cytoplasm. The differences in patterns of labeling among the various enzyme-gold complexes could be related to differences in affinities. The use of a panel of specific RNAses, displaying different affinities, could thus allow for the topographical distribution of particular RNA molecules according to their relative content of specific bases.

Animals↗
PubMed2003

Recombinant RNase Z does not recognize CCA as part of the tRNA and its cleavage efficieny is influenced by acceptor stem length.

One of the essential maturation steps to yield functional tRNA molecules is the removal of 3'-trailer sequences by RNase Z. After RNase Z cleavage the tRNA nucleotidyl transferase adds the CCA sequence to the tRNA 3'-terminus, thereby generating the mature tRNA. Here we investigated whether a terminal CCA triplet as 3'-trailer or embedded in a longer 3'-trailer influences cleavage site selection by RNase Z using three activities: a recombinant plant RNase Z, a recombinant archaeal RNase Z and an RNase Z active wheat extract. A trailer of only the CCA trinucleotide is left intact by the wheat extract RNase Z but is removed by the recombinant plant and archaeal enzymes. Thus the CCA triplet is not recognized by the RNase Z enzyme itself, but rather requires cofactors still present in the extract. In addition, we investigated the influence of acceptor stem length on cleavage by RNase Z using variants of wild-type tRNATyr. While the wild type and the variant with 8 base pairs in the acceptor stem were processed efficiently by all three activities, variants with shorter and longer acceptor stems were poor substrates or were not cleaved at all.

Arabidopsis↗
PubMed2001

In vitro function of S rnases in Lycopersicon peruvianum.

S RNases are products of the S locus that are expressed in the stylar tissue of Lycopersicon peruvianum with the gametophytic self-incompatibility (SI) system. Two S RNases (S12 and Sa) with RNase activity from the S12Sa genotype of L. peruvianum were purified using gel filtration and cation-exchange chromatography. The molecular masses of the two RNases, S12 and Sa, were 21 and 23.1 kDa, respectively. The specific activities of S12 and Sa for torula yeast rRNA as a substrate were 8,500 and 6,000 units/ml, respectively. Of various reagents tested for RNase activities, ZnSO4 and CuSO4 were found to remarkably reduce its activity. The growth of S12Sa pollen was inhibited when it was cultured in a pollen germination medium that contained the purified S12 RNase. The result suggested that the S RNase was either a probable inhibitor of pollen growth or controlled pollen growth. Additionally, 512Sa pollens germinated well in vitro in a germination medium that contained S12 RNase in the presence of ZnSO4 and CuSO4. Our finding suggests that the treatment of S RNase with its inhibitor destroys the SI ability on an in vitro self-pollen growth test.

Isoenzymes↗
PubMed1991

Bacteriophage T4 encodes an RNase H which removes RNA primers made by the T4 DNA replication system in vitro.

RNase H activity increases markedly after bacteriophage T4 infection of Escherichia coli MIC2003, an RNase H-deficient host. We have extensively purified the RNase H from these T4-infected cells and have shown that the RNase H activity copurifies with a 5' to 3' DNA exonuclease activity. The N-terminal sequence of a 35-kDa protein copurifying with the RNase H activity matches the terminus of the predicted product of an open reading frame (designated ORF A or 33.2) upstream of T4 gene 33, identified previously by Hahn and co-workers (Hahn, S., Kruse, U., and Rüger, W. (1986) Nucleic Acids Res. 14, 9311-9327). Plasmids containing ORF A under the control of the T7 promoter express RNase H and 5' to 3' DNA exonuclease activities as well as a protein that comigrates on sodium dodecyl sulfate-polyacrylamide gels with the 35-kDa protein present in the RNase H purified from T4-infected cells. T4 RNase H removes the pentamer RNA primers from DNA chains initiated by the T4 primase-helicase (gene products 61 and 41). Addition of T4 RNase H and T4 DNA ligase leads to extensive joining of discontinuous lagging strand fragments in the T4 DNA replication system in vitro.

Amino Acid Sequence↗
PubMed1991

Polyclonal antibodies against RNase L. Subcellular localization of this enzyme in mouse cells.

RNase L activated by 2-5A (a series of 2'-5'-linked adenylic oligoribonucleotides) is a key enzyme of the interferon system. To study RNase L (endonuclease L) in intact cells independently of intracellular 2-5A and of its activity, we have developed polyclonal antibodies against RNase L. RNase L from mouse spleen was purified on a column of 2-5A-Sepharose and used to immunize rabbits in co-injection with polyadenylic-polyuridylic acid as adjuvant. Antibodies were purified by chromatography on Affi-Gel blue and 2-5A-Sepharose-immobilized RNase L. These polyclonal antibodies immunoprecipitate the 80- and 40-kDa forms of RNase L in mouse spleen. In Western blot, only the 80-kDa form of RNase L is recognized by these antibodies. These purified antibodies were used to localize RNase L in the cytoplasm of intact mouse NIH 3T3 cells by immunofluorescence. The cytoplasmic localization of RNase L was confirmed by its 2-5A binding activity after cellular fractionation.

Animals↗
PubMed1995

Spinach chloroplast RNase P: a putative protein enzyme.

Ribonuclease P (RNase P) is the enzyme responsible for endonucleolytically separating the 5'-leader sequence from precursor tRNA molecules. In bacteria, and in the nuclei and mitochondria of all eukaryotes studied so far, RNase P contains an RNA subunit which is necessary for activity in vitro and in vivo. In contrast, we showed earlier that partially-purified RNase P from spinach chloroplasts had physical properties inconsistent with the presence of any RNA. We now report that the properties of the chloroplast enzyme, after 500 to 1500-fold purification, are consistent with enzymatic activity residing in a approximately 70 kDa polypeptide. Gel filtration chromatography on Sephacryl S-200 and S-300 provides a mass for chloroplast RNase P of approximately 70 +/- 5 kDa. A single polypeptide of approximately 70-80 kDa can be crosslinked to iodoUMP-substituted pre-tRNA. The labeling intensity of this polypeptide corresponds closely to the peak of RNase P activity on Sephacryl S-200 chromatography. Unlike the bacterial ribozyme-type RNase P, chloroplast RNase P is not a metalloenzyme. We showed previously that phosphodiester bond cleavage by the E. coli RNA enzyme absolutely requires Mg2+ or Mn2+ coordinated to the pro-Rp oxygen of the scissile phosphodiester phosphate. In contrast, we now find that chloroplast RNase P has no such requirement, and can accurately and efficiently cleave pre-tRNA containing an Rp-thio-substitution at the scissile bond. These data are entirely consistent with the hypothesis that RNase P in plant chloroplasts is not a ribozyme, but a conventional protein enzyme.

Biological Evolution↗
PubMed1996

RNase III autoregulation: structure and function of rncO, the posttranscriptional "operator".

Expression of the Escherichia coli rnc-era-recO operon is regulated posttranscriptionally by ribonuclease III (RNase III), encoded in the rnc gene. RNase III initiates rapid decay of the rnc operon mRNA by cleaving a double-stranded region of the rnc leader. This region, termed rncO, is portable, conferring stability and RNase III regulation to heterologous RNAs. Here, we report the detailed analysis of rncO structure and function. The first 215 nt of the rnc leader are sufficient for its function. Dimethylsulfate (DMS) modification in vivo revealed distinct structural elements in this region: a 13-nt single-stranded 5' leader, followed by a 6-bp stem-loop structure (I), a larger stem-loop structure (II) containing the RNase III site, a single-stranded region containing the rnc translation initiation site, and a small stem-loop structure (III) at the 3' terminus of rncO, wholly within the rnc coding region. Genetic analysis revealed the function of these structural elements. The single-stranded leader is not required for stability or RNase III control, stem-loop II is required only for RNase III control, and both stem-loops I and III are required for stability. Stem-loop II effectively serves only as the site at which RNase III cleaves to remove stem-loop I and thereby initiates decay, after which RNase III plays no role. Mutations at the cleavage site underscore the importance of base pairing for efficient RNase III attack. When stem-loops I and II were replaced with an artificial hairpin structure, stability was restored only partially, but was restored almost fully when a single-stranded leader was also added.

Base Sequence↗
PubMed1995

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.

Base Sequence↗
PubMed2002

Human endothelial cells selectively express large amounts of pancreatic-type ribonuclease (RNase 1).

Pyrimidine-specific ribonucleases are a superfamily of structurally related enzymes with distinct catalytic and biological properties. We used a combination of enzymatic and non-enzymatic assays to investigate the release of such enzymes by isolated cells in serum-free and serum-containing media. We found that human endothelial cells typically expressed large amounts of a pancreatic-type RNase that is related to, if not identical to, human pancreatic RNase. This enzyme exhibits pyrimidine-specific catalytic activity, with a marked preference for poly(C) substrate over poly(U) substrate. It was potently inhibited by placental RNase inhibitor, the selective pancreatic-type RNase inhibitor Inhibit-Ace, and a polyclonal antibody against human pancreatic RNase. The enzyme isolated from medium conditioned by immortalized umbilical vein endothelial cells (EA.hy926) possesses an amino-terminal sequence identical to that of pancreatic RNase, and shows molecular heterogeneity (molecular weights 18,000-26,000) due to different degrees of N-glycosylation. Endothelial cells from arteries, veins, and capillaries secreted up to 100 ng of this RNase daily per million cells, whereas levels were low or undetectable in media conditioned by other cell types examined. The corresponding messenger RNA was detected by RT-PCR in most cell types tested so far, and level of its expression was in keeping with the amounts of protein. The selective strong release of pancreatic-type RNase by endothelial cells suggests that it is endowed with non-digestive functions and involved in vascular homeostasis.

Amino Acid Sequence↗
PubMed1976

Effects of RNase and RNA on in vitro aster assembly.

RNase alters the in vitro assembly of spindle asters in homogenates of meiotically dividing surf clam (Spisula solidissima) oocytes. Some effects of RNase, such as reduced astral fiber length, appear nonenzymatic and probably result from RNase binding to tubulin. However, RNase-induced changes in the microtubule organizing center are also observed. Since other polycations can mimic RNase effects, the existence of an RNA component of the spindle organizing center remains uncertain. Effects of RNase and other polycations on astral fiber length can be prevented and reversed by the RNase inhibitor, polyguanylic acid. Polyguanylic acid can also augment astral fiber length in the absence of added RNase or other polycations. Augmentation by polyguanylic acid is favored by high ionic strength, and can be duplicated by polyuridylic acid and, with less efficiency, by polyadenylic acid. Polycytidylic acid and unfractionated yeast RNA, however, are unable to augment aster assembly. Polyguanylic acid can also augment the length of astral fibers on complete spindles isolated under polymerizing conditions. These results demonstrate that specific polyribonucleotides can alter spindle assembly in vitro. The presence of an inhibitor of microtubule assembly in Spisula oocytes, which can be inactivated by specific RNAs, is suggested.

Animals↗
PubMed1994

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor.

Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisin-catalyzed semisynthesis starting from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). The lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl-3',5'-adenosine and yeast RNA. When Lys-7 was replaced by S-methyl-cysteine or S-carboxamido-contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A-RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys-1, Cys-7-methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol-1. Modifications that introduced a positive charge in position 7 (S-aminoethyl- or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol-1 loss in binding energy. S-protein bound to RI with Ki = 63.4 pM, 800-fold weaker than RNase A. This corresponded to a 16-kJ mol-1 difference in binding energy. The results show that the N-terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.

Animals↗