Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.26.5 (RNase P)
 1,348 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

tRNA affinity chromatography, based on complex formation between tRNAs with complementary anticodons, has been applied to the isolation of specific tRNA precursors. When [32P]RNA, isolated from an Escherichia coli strain containing a thermolabile ribonuclease P, was chromatographed on resin-bound yeast phenylalanine tRNA, precursor tRNAGlu (possessing the complementary anticodon) was specifically retained. Likewise, precursor tRNAPhe was isolated from a column of resin-bound E. coli glutamate tRNA. Both precursor tRNAs isolated were monomeric and may be processed products of an originally larger RNA precursor. Both tRNA precursors contain additional nucleotides beyond the 5'-end of the mature tRNA and have all modified bases found in mature tRNA. The method can be extended to isolate other tRNA precursors by affinity chromatography with different tRNAs. Since the principle of complementary anticodon interaction is not restricted to any particular organism, specific precursor tRNAs from other sources may also be isolated in this way.
 ...
PMID:A method for the isolation of specific tRNA precursors. 110 1

Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that removes precursor sequences from 5'-ends of pre-tRNAs. A photoaffinity cross-linking agent was coupled to the substrate phosphate on which RNase P acts and used to map nucleotides in the vicinity of the catalytic site of this ribozyme. Mature tRNA(Phe) containing a 5'-thiophosphate was synthesized by transcription in vitro using phage T7 RNA polymerase in the presence of guanosine 5'-phosphorothioate. The photoagent (azidophenacyl) was coupled uniquely to the 5'-thiophosphate of the tRNA, the site of action by RNase P. The photoagent-containing tRNA binds to RNase P RNA and is cross-linked by UV irradiation to it at high efficiency (10-30%). Cross-linked conjugates are enzymatically inactive, consistent with the occupancy of the active site of the RNase P RNA by the tRNA. Reversal of the cross-link by phenylmercuric acetate restores activity. The sites of cross-linking in RNase P RNA were determined by primer extension. In order to identify generalities and detect idiosyncrasies, analyses were carried out using RNase P RNAs from three phylogenetically diverse organisms: Bacillus subtilis, Chromatium vinosum and Escherichia coli. In the context of a phylogenetic structure model, two regions of cross-linking are observed in all three RNAs. Two of the RNAs cross-link to a lesser extent at a third structural region and one of the RNAs is cross-linked to a small extent to a fourth region. All the sites of cross-linking between the substrate phosphate in tRNA and the RNase P RNAs are in the conserved core of the structure model, consistent with the importance of the cross-linked residues to the action of this RNA enzyme.
 ...
PMID:Mapping the active site of ribonuclease P RNA using a substrate containing a photoaffinity agent. 170 Nov 42

A synthetic tRNA precursor analog containing the structural elements of Escherichia coli tRNA(Phe) was characterized as a substrate for E. coli ribonuclease P and for M1 RNA, the catalytic RNA subunit. Processing of the synthetic precursor exhibited a Mg2+ dependence quite similar to that of natural tRNA precursors such as E. coli tRNA(Tyr) precursor. It was found that Sr2+, Ca2+, and Ba2+ ions promoted processing of the dimeric precursor at Mg2+ concentrations otherwise insufficient to support processing; very similar behavior was noted for E. coli tRNA(Tyr). As noted previously for natural tRNA precursors, the absence of the 3'-terminal CA sequence in the synthetic precursor diminished the facility of processing of this substrate by RNase P and M1 RNA. A study of the Mg2+ dependence of processing of the synthetic tRNA dimeric substrate radiolabeled between C75 and A76 provided unequivocal evidence for an alteration in the actual site of processing by E. coli RNase P as a function of Mg2+ concentration. This property was subsequently demonstrated to obtain (Carter, B. J., Vold, B.S., and Hecht, S. M. (1990) J. Biol. Chem. 265, 7100-7103) for a mutant Bacillus subtilis tRNAHis precursor containing a potential A-C base pair at the end of the acceptor stem.
 ...
PMID:Metal ion and substrate structure dependence of the processing of tRNA precursors by RNase P and M1 RNA. 226 41

Two distinct RNase P-like activities which cleave leader sequences from pre-tRNA molecules to give mature 5' ends have been identified in carrot suspension-culture cells. An Escherichia coli pre-tRNA(Phe) and a tobacco pre-tRNA(Tyr) were transcribed in vitro then used as substrates for processing reactions in a cell-free extract. The pre-tRNA(Tyr) transcript was used to establish optimal salt and divalent cation requirements for processing. Kinetic experiments were then carried out on both substrates to determine if 5' and 3' processing were ordered. Primer extension analysis of processing intermediates and stable products verified that an ammonium sulfate fraction of the extract was indeed capable of accurately processing the 5' ends of both pre-tRNAs. Subsequent fractionation of the 5' end-processing activity by chromatography on phosphocellulose revealed two distinct activities, eluting at 0.1 and 0.5 M KCI, when assayed with the tobacco pre-tRNA(Tyr) substrate. When the same fractions were assayed with the E. coli pre-tRNA(Phe), only the 0.1 M KCI fraction exhibited activity. Both of the active fraction display sensitivity to micrococcal nuclease (MN) and proteinase K indicating each is a ribonucleoprotein, a result not seen with other plant RNase Ps. Subsequent FPLC fractionation of the two activities using Mono Q and Mono S columns demonstrated that the two activities could be further distinguished on the basis of their chromatographic behavior.
 ...
PMID:Characterization and partial purification of two pre-tRNA 5'-processing activities from Daucus carrota (carrot) suspension cells. 774 55

A new approach for modification interference studies is presented. It involves the use of phosphorothioates as a handle to analyze any desired base or sugar modification. This method was applied to identify ribose and phosphate moieties which could be important in the pre-tRNA recognition of E. coli RNase P RNA (M1 RNA). The utility of this technique was confirmed by detecting the inhibitory effect of a deoxyribose in the 5'-flank (position-1). This site was already known to interfere with RNase P cleavage, if modified. We have analyzed pre-tRNA(Tyr) and pre-tRNA(Phe) and found different interference patterns for both tRNAs. Two unpaired regions were involved in both pre-tRNAs. Phosphorothioates interfered at the transition between acceptor- and D-arms. The results with deoxythymidines in the T-loop indicated that deoxyribose moieties or the extra methyl group in thymidine could interfere with RNAse P cleavage. These data suggest that even in complete pre-tRNAs, only a few intact ribonucleotides are important in the substrate recognition by RNase P. We have demonstrated the potential of this new approach which offers many future applications in all fields involving nucleic acids, for example RNA processing, action of ribozymes, tRNA charging and studies related to DNA promoter recognition.
 ...
PMID:Modification interference approach to detect ribose moieties important for the optimal activity of a ribozyme. 844 16

We suggested previously that a purine at the discriminator base position in a tRNA precursor interacts with the well-conserved U294 in M1 RNA, the catalytic subunit of Escherichia coli RNase P. Here we investigated this interaction and its influence on the kinetics of cleavage as well as on cleavage site selection. The discriminator base in precursors to tRNA(Tyr)Su3 and tRNA(Phe) was changed from A to C and cleavage kinetics were studied by wild-type M1 RNA and a mutant M1 RNA carrying the compensatory substitution of a U to a G at position 294 in M1 RNA. Our data suggest that the discriminator base interacts with the residue at position 294 in M1 RNA. Although product release is a rate-limiting step both in the absence and in the presence of this interaction, its presence results in a significant reduction in the rate of product release. In addition, we studied cleavage site selection on various tRNA(His) precursor derivatives. These precursors carry a C at the discriminator base position. The results showed that the mutant M1 RNA harboring a G at position 294 miscleaved a wild-type tRNA(His) precursor and a tRNA(His) precursor carrying an inosine at the cleavage site. The combined data suggest a functional interaction between the discriminator base and the well-conserved U294 in M1 RNA. This interaction is suggested to play an important role in determining the rate of product release during multiple turnover cleavage of tRNA precursors by M1 RNA as well as in cleavage site selection.
 ...
PMID:Interaction between Escherichia coli RNase P RNA and the discriminator base results in slow product release. 863 10

The phosphorothioate footprinting technique was applied to the investigation of phosphate moieties in tRNA substrates involved in interactions with M1 RNA, the catalytic subunit of Escherichia coli RNase P. In general agreement with previous data, all affected sites were localized in acceptor stem and T arm. But the analyzed examples for class I (Saccharomyces cerevisiae pre-tRNA(Phe) with short variable arm) and class II tRNAs (E. coli pre-tRNA(Tyr) with large variable arm) revealed substantial differences. In the complex with pre-tRNA(Phe), protection was observed at U55, C56, and G57, along the top of the T loop in the tertiary structure, whereas in pre-tRNA(Tyr), the protected positions were G57, A58, and A59, at the bottom of the T loop. These differences suggest that the size of the variable arm affects the spatial arrangement of the T arm, providing a possible explanation for the discrepancy in reports about the D arm requirement in truncated tRNA substrates for eukaryotic RNase P enzymes. Enhanced reactivities were found near the junction of acceptor and T stem (U6, 7, 8 in pre-tRNA(Phe) and G7, U63, U64 in pre-tRNA(Tyr)). This indicates a partial unfolding of the tRNA structure upon complex formation with RNase P RNA.
 ...
PMID:Differences in the interaction of Escherichia coli RNase P RNA with tRNAs containing a short or a long extra arm. 875 10

The ribozyme from bacterial ribonuclease P (denoted P RNA) specifically recognizes the coaxially stacked T stem-loop and the acceptor stem of a tRNA substrate. This recognition is mediated primarily through tertiary interactions. At least four 2'-OH groups in the T stem-loop region have been implicated as direct contacts with Bacillus subtilis P RNA [Pan, T., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12510]. Effects of six single 2'-OH --> 2'-H substitutions and two base mutants of the G19-C56 tertiary interaction in tRNA on substrate binding (Kd) and the chemical step of the reaction (k2) have been determined using a tRNA(Phe) substrate containing a 2'-deoxy residue at the cleavage site. Our results show that at least five functional groups in the T stem-loop of tRNA directly participate in P RNA binding. They include the 2'-OH groups of residues 54, 56, 61, and 62 and possibly the 4-amino group of the conserved C56. The 2'-OHs of residues 54, 61, and 62 are positioned within the same minor groove due to stacking of the reverse Hoogsteen U54-A58 pair on the G53-C61 Watson-Crick pair in the T stem. This groove is extended to the 4-amino group of C56 through the tertiary structure of tRNA. We use the term "tertiary groove" to describe alignment of functional groups through tertiary folding of an RNA. The binding also includes the 2'-OH of nucleotide C56 which is not located in this tertiary groove. Assuming additivity, these five interactions can contribute 7.4 kcal/mol or 10(5)-fold in binding but only -0.5 kcal/mol or approximately 2-fold in chemistry at 37 degrees C. The P RNA binding site for the T stem-loop includes at least the previously identified A230 as well as the A130 in B. subtilis P RNA. The Kd and k2 data from the A130G mutant of B. subtilis P RNA suggest that A130 may be proximal to residue 56 in tRNA. These results show how the highly structured T stem-loop region in a pre-tRNA substrate is bound by the B. subtilis P RNA. This is among the first examples of how a nonhelical RNA structure can be recognized by another RNA through tertiary interactions.
 ...
PMID:Recognition of the T stem-loop of a pre-tRNA substrate by the ribozyme from Bacillus subtilis ribonuclease P. 917 46

The Saccharomyces cerevisiae nuclear gene RPM2 encodes a component of the mitochondrial tRNA-processing enzyme RNase P. Cells grown on fermentable carbon sources do not require mitochondrial tRNA processing activity, but still require RPM2, indicating an additional function for the Rpm2 protein. RPM2-null cells arrest after 25 generations on fermentable media. Spontaneous mutations that suppress arrest occur with a frequency of approximately 9 x 10(-6). The resultant mutants do not grow on nonfermentable carbon sources. We identified two loci responsible for this suppression, which encode proteins that influence proteasome function or assembly. PRE4 is an essential gene encoding the beta-7 subunit of the 20S proteasome core. A Val-to-Phe substitution within a highly conserved region of Pre4p that disrupts proteasome function suppresses the growth arrest of RPM2-null cells on fermentable media. The other locus, UMP1, encodes a chaperone involved in 20S proteasome assembly. A nonsense mutation in UMP1 also disrupts proteasome function and suppresses Deltarpm2 growth arrest. In an RPM2 wild-type background, pre4-2 and ump1-2 strains fail to grow at restrictive temperatures on nonfermentable carbon sources. These data link proteasome activity with Rpm2p and mitochondrial function.
 ...
PMID:Proteasome mutants, pre4-2 and ump1-2, suppress the essential function but not the mitochondrial RNase P function of the Saccharomyces cerevisiae gene RPM2. 1075 50

We apply synchrotron-based small-angle X-ray scattering to investigate the relationship between compaction, metal binding, and structure formation of two RNAs at 37 degrees C: the 76 nucleotide yeast tRNA(Phe) and the 255 nucleotide catalytic domain of the Bacillus subtilis RNase P RNA. For both RNAs, this method provides direct evidence for the population of a distinct folding intermediate. The relative compaction between the intermediate and the native state does not correlate with the size of the RNA but does correlate well with the amount of surface burial as quantified previously by the urea-dependent m-value. The total compaction process can be described in two major stages. Starting from a completely unfolded state (4-8 M urea, no Mg(2+)), the major amount of compaction occurs upon the dilution of the denaturant and the addition of micromolar amounts of Mg(2+) to form the intermediate. The native state forms in a single transition from the intermediate state upon cooperative binding of three to four Mg(2+) ions. The characterization of this intermediate by small-angle X-ray scattering lends strong support for the cooperative Mg(2+)-binding model to describe the stability of a tertiary RNA.
 ...
PMID:Mg2+-dependent compaction and folding of yeast tRNAPhe and the catalytic domain of the B. subtilis RNase P RNA determined by small-angle X-ray scattering. 1099 49


1 2 Next >>