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PubMed1985

Glycation of amino groups in protein. Studies on the specificity of modification of RNase by glucose.

Ribonuclease A has been used as a model protein for studying the specificity of glycation of amino groups in protein under physiological conditions (phosphate buffer, pH 7.4, 37 degrees C). Incubation of RNase with glucose led to an enhanced rate of inactivation of the enzyme relative to the rate of modification of lysine residues, suggesting preferential modification of active site lysine residues. Sites of glycation of RNase were identified by amino acid analysis of tryptic peptides isolated by reverse-phase high pressure liquid chromatography and phenylboronate affinity chromatography. Schiff base adducts were trapped with Na-BH3CN and the alpha-amino group of Lys-1 was identified as the primary site (80-90%) of initial Schiff base formation on RNase. In contrast, Lys-41 and Lys-7 in the active site accounted for about 38 and 29%, respectively, of ketoamine adducts formed via the Amadori rearrangement. Other sites reactive in ketoamine formation included N alpha-Lys-1 (15%), N epsilon-Lys-1 (9%), and Lys-37 (9%) which are adjacent to acidic amino acids. The remaining six lysine residues in RNase, which are located on the surface of the protein, were relatively inactive in forming either the Schiff base or Amadori adduct. Both the equilibrium Schiff base concentration and the rate of the Amadori rearrangement at each site were found to be important in determining the specificity of glycation of RNase.

Amino Acid Sequence↗
PubMed1980

RNase P of Bacillus subtilis has a RNA component.

Ribonuclease P from Bacillus subtilis cleaves a Gln-Leu tRNA dimeric precursor from bacteriophage T4-infected Escherichia coli, yielding products identical with those generated by the E. coli RNase P. Using this tRNA dimer as an assay substrate, the RNase of P of B. subtilis was shown to consist of at least two components, one of which bands in CsCl equilibrium buoyant density centrifugation at 1.7 g/ml, characteristic of a protein x nucleic acid complex. Both this component and a second, retrieved from the low density (less than 1.4 g/ml) regions of CsCl gradients, are required for RNase P activity. Enzyme activity is abolished by treating the component of density 1.7 g/ml with insoluble RNase A prior to assay. These observations suggest that the RNase P of B. subtilis, like that of E. coli, contain a RNA component essential for activity. That this RNA component is of functional importance, and not an artifact of isolation procedures, is supported by the fact that it is observed in these two phylogenetically disparate organisms.

Bacillus subtilis↗
PubMed1984

A site in a tRNA precursor that can be processed by the whole RNase P enzyme but not by the RNA alone.

A precursor molecule for 10 Sb RNA, the RNA moiety of the RNA processing enzyme RNase P, was purified, characterized for enzymatic activity, and compared to 10 Sb RNA and to RNase P. In these studies the K RNA, a dimeric precursor of tRNAGln-tRNALeu, coded by bacteriophage T4, was used as a substrate. This precursor contains two RNase P cleavage sites, one at each 5' end of the two tRNAs. The precursor 10 Sb and 10 Sb RNAs have the capacity to cleave the precursor tRNA molecule but only at the 5' end of tRNALeu, not at the 5' end of tRNAGln. Even when a substrate was prepared that contained only one site for RNase P (the one next to tRNAGln), this substrate was not cleaved by the RNA alone while the whole enzyme was effective in processing this substrate. The possible function of the protein of RNase P in the enzymatic reaction is discussed.

Base Sequence↗
PubMed1981

Partial characterization of ribonuclease (RNase) activity from the isolated and solubilized brush border of Hymenolepis diminuta.

The isolated brush border membrane of Hymenolepis diminuta contained ribonuclease (RNase) activity which was demonstrable using yeast RNA or synthetic homopolymers of adenylic, cytidylic, inosinic, or uridylic acids as substrates. Polyguanylic acid was not hydrolyzed by worm RNase. RNase activity was inhibited by EDTA and divalent cations as well as sulfhydryl blocking and reducing agents. Polyguanylic acid and DNA were also inhibitors of RNase activity; these compounds were not hydrolyzed, but inhibited the hydrolysis of other substrates, possibly by nonproductive substrate binding. Data suggested that RNase (endonuclease) was probably the major enzyme activity in the degradation of long chain polyribonucleotides at the work's surface, while phosphodiesterase (exonuclease) activity did not contribute significantly to the hydrolysis of these compounds.

Animals↗
PubMed1994

Effects of nucleotide sequence on the specificity of rne-dependent and RNase E-mediated cleavages of RNA I encoded by the pBR322 plasmid.

RNase E, an endoribonuclease encoded by the Escherichia coli ams/rne/hmp1 locus, cleaves RNA I, an antisense regulator of the replication of ColE1 type plasmids, in a single-stranded region near its 5' end. The rne-3071 mutation prolongs the RNA 1 half-life in cells cultured at an elevated temperature and imparts temperature sensitivity on RNase E isolated from the mutant strain. Here we report the effects of specific sequence changes introduced by site-directed mutagenesis on the location of ribonucleolytic cleavage near the 5' end of pBR322 RNA I in rne-3071 and congenic rne+ E. coli and on cleavage of RNA I by RNase E in vitro. Primer extension analyses showed that the occurrence and position of cleavages in vivo and in vitro are altered highly specifically by sequence changes but that the site of cleavage bears no simple relationship to a particular nucleotide order. Our results do not support either the notion that cleavage by RNase E is determined by a consensus sequence or the contrary view that RNase E is a virtually nonspecific single-stranded endonuclease with a preference for cutting 5' to an AU dinucleotide.

Base Sequence↗
PubMed1994

Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase.

2-5A-dependent RNase (RNase L), a unique endoribonuclease that requires 5'-phosphorylated 2',5'-linked oligoadenylates (2-5A), functions in the molecular mechanism of interferon action. Because this enzyme is present at very low levels in nature, characterization and analysis have been limited. The molecular cloning of human, 2-5A-dependent RNase cDNA has facilitated its expression to high levels in insect cells by infecting with recombinant baculovirus. To determine the properties of the enzyme in the absence of other proteins, the recombinant 2-5A-dependent RNase was purified to homogeneity. The purified enzyme migrated as a monomer upon gel filtration in the absence of activator and showed highly specific, 2-5A-dependent RNase activity. The precise activator requirements were determined by stimulating the purified enzyme with a variety of 2',5'-linked oligonucleotides. The activated enzyme was capable of cleaving poly(rU) and, to a lesser extent, poly(rA), to sets of discrete products ranging from between 4 and 22 nucleotides in length. Reduced rates of 2-5A-dependent RNA cleavage were observed even after removal of ATP and chelation of divalent cations. However, optimal RNA cleavage rates required the presence of either manganese or magnesium and ATP.

Adenosine Triphosphate↗
PubMed1993

RNase H activity of reverse transcriptases on substrates derived from the 5' end of retroviral genome.

RNA/DNA substrates derived from the 5' ends of human immunodeficiency virus (HIV) and Moloney murine leukemia virus (MMuLV) genomes were used to study the specificity of the RNase H activities of HIV, AMV (avian myeloblastosis virus), and MMuLV reverse transcriptases. These substrates were selected because they represent the site for the first template switch during proviral DNA synthesis. Variability of cleavage was observed depending on the origin of the enzyme as well as the sequence of the RNA/DNA substrate. The minimal size of hybrid recognized by the RNase H activity of reverse transcriptase was also affected by the same parameters, namely, the enzyme and the substrate origin. Moreover, the size of the residual 5'-undigested RNA after completion of the RNase H reaction depended on the position of the DNA annealed to the genomic RNA. When the hybrid was located at the 5' R region of the viral genome, stable hybrids with RNAs of 13-18 nucleotides remained following digestion by HIV reverse transcriptase, and 21-24 nucleotides following digestion by AMV reverse transcriptase and MMuLV reverse transcriptase. On the other hand, with all three enzymes, smaller sized hybrids remained when the DNA was hybridized to internal U5 or R sequences. The reason for this variance in size appears to be the inability of RNase H to efficiently digest at the 5' end of hybrid structures. Surprisingly, hybridization to the RNA template, of a DNA oligomer that extended 15 nucleotides beyond the 5' end of the RNA R region sequences, resulted in further digestion of the RNA. This unexpected mode of action of RNase H at the 5' end of the genomic RNA should be taken in consideration in studies of the first template switch.

Avian Myeloblastosis Virus↗
PubMedDate not supplied

RNase L and increased endoribonuclease activities in the mononuclear cells of patients with chronic myelogenous leukemia.

During investigations of the interferon-induced 2',5' oligoadenylate synthetase/RNase L system in malignancy, RNase L activity and an increased endoribonuclease activity were observed in peripheral blood mononuclear cell (PBMC) extracts from patients with chronic myelogenous leukemia. The cleavage of rRNA from intact ribosomes was used as the assay for both RNase L and the increased endoribonuclease activities. Novel rRNA cleavage products (NCP) were generated by extracts of Ficoll-purified mononuclear cells from chronic myelogenous leukemia (CML) patients and in the granulocytic fraction of both patients and healthy controls. Determination of the time course of rRNA degradation demonstrated that the novel cleavage products were rapidly derived from the further endoribonucleolytic degradation of the RNase L derived specific cleavage products. Prolonged incubation of mononuclear cell extracts from healthy controls also yielded the novel rRNA cleavage products. Comparisons of the kinetics of NCP production suggest that the novel endoribonuclease activity can be approximately 240-fold greater in PBMC extracts from CML patients than controls. Analysis of peripheral blood WBC count and differential indicated that the increased RNase activities were associated with the presence of immature granulocytic cells in the peripheral blood (p = 0.001, Fisher's exact test). However, these activities were also found in the mononuclear cells of a CML patient in lymphoid blast crisis. Since CML is a stem cell disease, the novel endoribonuclease activity may be indicative of active disease, rather than a marker for immature granulocytes. Thus, the RNase L and increased endoribonuclease activities may play a functional role in the biology of chronic myelogenous leukemia and may be important in the mechanism of action of interferon therapy in this disease.

Endoribonucleases↗
PubMedDate not supplied

[DNases and RNases of Misgurnus fossilis ovocytes].

The pH optima were determined for DNases and RNases of the loach eggs. For DNases they are 5.6 and 7.6 and for RNases - 5.2 and 7.2. It is established that Ca++ activates, and Fe++ has not effect on the activity of acid and alkaline DNases, while Mg++, Mn++ and especially Co++, Zn++, Cd++, Cu++ have an inhibitory effect on them. The activities of RNases is stimulated by Ca++ and Fe++, and inhibited by Zn++, Co++, Cd++ and Cu++. Iones Mg++ and Mn++ do not affect these activities. Localization of the above mentioned enzymes was studied by means of differential centrifugation of egg homogenates. Acid DNase is concentrated only in postmicrosomal supernatant liquid, its activity being inhibited in the presence of the nucleomitochondrial and microsomal fractions. Acid RNase is also localized predominantly in postmicrosomal supernatant fraction. Alkaline DNase is found to a great extent in nucleomitochondrial fraction, and alkaline RNase - in postmicrosomal one.

Animals↗
PubMed1993

Yeast mitochondrial RNase P. Sequence of the RPM2 gene and demonstration that its product is a protein subunit of the enzyme.

We report here the sequence of the RPM2 gene which codes for the 105-kDa protein previously purified from the mitochondria of Saccharomyces cerevisiae and shown by genetic techniques to be required for mitochondrial RNase P activity. The sequence predicts a primary translation product of 1202 residues with a molecular mass of 139 kDa and no obvious sequence similarity to any known protein in the data bases. There are 122 amino-terminal amino acids predicted by the gene that are not found in the purified protein, some of which may play a role in mitochondrial targeting of the protein. Antibodies raised against a trpE-105-kDa fusion protein recognize a 105-kDa protein in wild-type cells but not in cells carrying a disruption of the RMP2 gene. Immune, but not preimmune serum, immunoprecipitates the RNase P RNA and the mitochondrial RNase P activity. Thus, the 105-kDa protein forms a complex with RNase P RNA and is required for RNase P activity as predicted for a bona fide subunit of the enzyme.

Amino Acid Sequence↗
PubMed1996

Phylogenetic comparative mutational analysis of the base-pairing between RNase P RNA and its substrate.

We have studied the base-pairing between the 3'-terminal CCA motif of a tRNA precursor and RNase P RNA by a phylogenetic mutational comparative approach. Thus, various derivatives of the Escherichia coli tRNA(Ser)Su1 precursor harboring all possible substitutions at either the first or the second C of the 3'-terminal CCA motif were generated. Cleavage site selection on these precursors was studied using mutant variants of M1 RNA, the catalytic subunit of E. coli RNase P, carrying changes at positions 292 or 293, which are involved in the interaction with the 3'-terminal CCA motif. From our data we conclude that these two C's in the substrate interact with the well-conserved G292 and G293 through canonical Watson-Crick base-pairing. Cleavage performed using reconstituted holoenzyme complexes suggests that this interaction also occurs in the presence of the C5 protein. Furthermore, we studied the interaction using various derivatives of RNase P RNAs from Mycoplasma hyopneumoniae and Mycobacterium tuberculosis. Our results suggest that the base-pairing between the 3'-terminal CCA motif and RNase P is present also in other bacterial RNase P-substrate complexes and is not limited to a particular bacterial species.

Bacterial Proteins↗
PubMed1996

Slow folding kinetics of RNase P RNA.

Understanding the folding mechanisms of large, highly structured RNAs is important for understanding how these molecules carry out their function. Although models for the three-dimensional architecture of several large RNAs have been constructed, the process by which these structures are formed is only now beginning to be explored. The kinetic folding pathway of the Tetrahymena ribozyme involves multiple intermediates and both Mg2+-dependent and Mg2+-independent steps. To determine whether this general mechanism is representative of folding of other large RNAs, a study of RNase P RNA folding was undertaken. We show, using a kinetic oligonucleotide hybridization assay, that there is at least one slow step on the folding pathway of RNase P RNA, resulting in conformational changes in the P7 helix region on the minute timescale. Although this folding event requires the presence of Mg2+, the slow step itself does not involve Mg2+ binding. The P7 and P2 helix regions exhibit distinctly different folding behavior and ion dependence, implying that RNase P folding is likely to be a complex process. Furthermore, there are distinct similarities in the folding of RNase P RNA from both Bacillus subtilis and Escherichia coli, indicating that the folding pathway may also be conserved along with the final structure. The slow folding kinetics, Mg2+-independence of the rate, and existence of intermediates are basic features of the folding mechanism of the Tetrahymena group I intron that are also found in RNase P RNA, suggesting these may be general features of the folding of large RNAs.

Animals↗
PubMed1996

Rp-deoxy-phosphorothioate modification interference experiments identify 2'-OH groups in RNase P RNA that are crucial to tRNA binding.

Ribose 2'-hydroxyls make a key contribution to the enormous structural and functional potential of RNA molecules. Here, we report the identification of 2'-deoxy modifications in the catalytic RNA subunit of RNase P from Escherichia coli that interfere with tRNA binding. This was accomplished by modification interference employing pools of RNase P RNA that carried a low level of Rp-deoxy-phosphorothioate (Rp-deoxyNMPalpha(S) ) modifications randomly distributed over its 380 nt. A gel retardation assay allowed us to separate RNase P RNA pools into tRNA-binding and nonbinding fractions. Differences in the intensity of phosphorothioate-specific iodine hydrolysis patterns of the two RNA fractions revealed positions where the Rp-deoxyNMPalpha(S) modification interferes with tRNA binding. A comparison with interference patterns obtained for the Rp-NMPalpha(S) modification alone has identified some 20 positions in the backbone of E. coli RNase P RNA where the functional defect caused by the Rp-deoxyNMPalpha(S) double modification is attributable to the 2'-deoxy modification (or possibly the C5 methyl group in the case of U residues because we used deoxyTMPalpha(S) for partial substitution of UMP). Most of the corresponding 2'-OH functions were localized in regions that have been reported to crosslink to photoreactive tRNA derivatives, suggesting that these 2'-hydroxyls are located along the tRNA binding interface of E. coli RNase P RNA. Our results indicate that the modification interference approach applied here will be useful generally to identify structurally and functionally important 2'-hydroxyls in large RNAs and ribozymes.

Animals↗
PubMed1978

Characterization of an endoribonuclease, RNase N, from Escherichia coli.

The properties of the enzyme ribonuclease N were investigated. By comparing the distribution in the cell of RNase N with the bonafide intracellular beta-galactosidase, and the periplasmic alkaline phosphatase enzymes, we showed that RNase N is an intracellular enzyme. Since previous studies suggested that it is an endoribonuclease, it was compared to RNase III, the only other known intracellular endoribonuclease in Escherichia coli. Using homopolymers and co-polymers we found that, while RNase III could digest double-stranded RNA only, RNase N digested single-stranded and double-stranded RNA with similar efficiency. Furthermore, all RNAs used, natural as well as synthetic, were substrates for the enzyme. Using 5 S rRNA as a substrate it was confirmed that the enzyme is an endonuclease. The final products of the reaction of this enzyme are 5'-mononucleotides. The molecular weight of the enzyme is about 120,000 and it seems to contain two subunits which are similar in size. These properties thus differentiate this enzyme from all other known ribonucleases in E. coli.

Alkaline Phosphatase↗
PubMed1999

The crystal structure of recombinant rat pancreatic RNase A.

The three-dimensional structure of rat pancreatic RNase A expressed in Escherichia coli was determined. The backbone conformations of certain critical loops are significantly different in this enzyme compared to its bovine counterpart. However, the core structure of rat RNase A is similar to that of the other members of the pancreatic ribonuclease family. The structural variations within a loop bordering the active site can be correlated with the subtle differences in the enzymatic activities of bovine and rat ribonucleases for different substrates. The most significant difference in the backbone conformation was observed in the loop 15-25. This loop incorporates the subtilisin cleavage site which is responsible for RNase A to RNase S conversion in the bovine enzyme. The rat enzyme does not get cleaved under identical conditions. Molecular docking of this region of the rat enzyme in the active site of subtilisin shows steric incompatibility, although the bovine pancreatic ribonuclease A appropriately fits into this active site. It is therefore inferred that the local conformation of the substrate governs the specificity of subtilisin.

Amino Acid Sequence↗
PubMed2004

Addressing the challenge of changing the specificity of RNase T1 with rational and evolutionary approaches.

Although ribonuclease T1 (RNase T1) is one of the best-characterized proteins with respect to structure and enzymatic action, numerous attempts at altering the specificity of the enzyme to cleave single-stranded RNA at the 3'-side of adenylic instead of guanylic residues by rational approaches have failed so far. Recently we generated and characterized the RNase T1 variant RV with a 7200-fold increase in adenylyl-3',5'-cytidine (ApC)/guanylyl-3',5'-cytidine (GpC) preference, with the guanine-binding loop changed from 41-KYNNYE-46 (wt) to 41-EFRNWN-46. Now we have introduced the asparagine residue at position 46 of the wild-type enzyme as a single-point mutation in variant E46N and in combination with the Y45W exchange also occurring in RV. Both variants show an improved ApC/GpC preference with a 1450-fold increase for E46N and a 2100-fold increase for Y45W/E46N in comparison to wild-type activity. We also addressed the challenge of altering enzyme specificity with an evolutionary approach. We have randomly introduced point mutations into the RNase T1 wild-type gene and into the gene of the variant RV with different mutation rates. Altogether we have screened about 100,000 individual clones for activity on RNase indicator plates; 533 of these clones were active. A significant change in substrate specificity towards an ApC preference could not be observed for any of these active variants; this demonstrated the magnitude of the challenge to alter the specificity of this evolutionary perfected enzyme.

Amino Acids↗
PubMed1988

A novel function of RNase P from Escherichia coli: processing of a suppressor tRNA precursor.

The leuX gene of Escherichia coli codes for a suppressor tRNA and forms a single gene operon containing its own promoter and Q-independent terminator. An analysis of the in vitro processing of leuX precursor revealed that the processing of the 5' end took place in a single-step reaction catalysed by RNase P while the 3' processing involved two successive reactions. The endonucleolytic cleavage activity of the 3' precursor sequence was found to copurify with RNase P. Heat inactivation of thermosensitive RNase P from two independent E. coli mutants abolished the cleavage activity of both the 5' and 3' ends. These results altogether suggest that RNase P carries the activity of 3' end cleavage as well as that of 5' processing. In the presence of Mg2+ alone, the leuX precursor was found to be self-cleaved at a site approximately 13 nt inside from the 5' end of mature tRNA. The self-cleaved precursor tRNA was no longer processed by the 3' endonuclease, suggesting that the 3' endonuclease recognizes a specific conformation of the precursor tRNA for action.

Base Sequence↗
PubMed1990

Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III.

The replication frequency of IncFII plasmids is regulated through the availability of a rate-limiting protein, RepA. The synthesis of this protein is controlled post-transcriptionally by a small antisense RNA, CopA, which binds to the leader region of the RepA mRNA (CopT). In this communication we report studies of the IncFII plasmid R1. We show that the duplex between CopA and CopT is cleaved specifically in vivo. The in vivo cleavage maps to the same position as that resulting from in vitro cleavage of a CopA/CopT duplex by purified RNase III. By introducing plasmids carrying translational repA-lacZ fusions into cells deficient in RNase III we show that the expression of repA is elevated when RNase III activity is severely decreased. Hence, cleavage by RNase III seems to be a key event in the copy number control system of plasmid R1.

Bacterial Proteins↗