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

A precursor molecule to the stable 4.5 S RNA species of Escherichia coli has been found to accumulate at 42 degrees in a strain thermosensitive for the function of ribonuclease P. The precursor molecule is 130 nucleotides long. Twenty-two extra nucleotides, starting with pppGp, precede the mature sequence at its 5' terminus. At least 1 extra uridine residue can be found at the 3' terminus. The precursor to 4.5 S RNA is cleaved in vitro by RNase P to generate a 5' end identical to that of the mature 4.5 S RNA.
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PMID:Nucleotide sequence and in vitro processing of a precursor molecule to Escherichia coli 4.5 S RNA. 79 64

4-Thiouridine (s4U), a photoreactive analog of uridine, was randomly incorporated into tRNA2(fMet) precursor molecules by transcription with T7 RNA polymerase. The s4U-containing transcripts were trimmed at their 5'-ends with RNase P RNA to yield mature tRNA2(fMet). The photoreactive tRNA2(fMet) derivatives were aminoacylated and bound to the P site of 70S ribosomes from Escherichia coli in the presence of a poly(A,G,U) template. Irradiation of the complexes at 300 nm resulted in the covalent cross-linking of tRNA2(fMet) to ribosomal proteins and rRNAs within both the 50S and 30S subunits. The labeled proteins were identified as L1, L27, and S19. 50S-subunit proteins L1 and L27 were attached to nucleotide U17 or U17.1 within the D loop of tRNA2(fMet), whereas 30S-subunit protein S19 was cross-linked to nucleotide U47 in the variable loop. Both of these sites occur in or near the central fold of the tRNA. These results permit us to map the D loop of P site-bound tRNA to the region between the central protuberance and the L1 ridge on the 50S ribosomal subunit, while the variable loop can be placed above the cleft on the head of the 30S subunit.
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PMID:Mapping the central fold of tRNA2(fMet) in the P site of the Escherichia coli ribosome. 825 1

4-Thiouridine, a photoreactive analogue of uridine, was randomly incorporated into yeast tRNA(Phe) precursor molecules by transcription with T7 RNA polymerase and the resulting transcripts were converted into mature tRNA(Phe) by treatment with RNase P RNA. The photoreactive tRNA(Phe) was aminoacylated and bound to the P site of Escherichia coli 70S ribosomes in the presence of a poly(U) template. Irradiation of the complexes with light of 300 nm resulted in the covalent crosslinking of nt U20 in the D loop of the tRNA to protein S11 of the 30S ribosomal subunit, whereas nt U33 in the anticodon loop crosslinked to 30S-subunit protein S7. These results allowed us to map the D loop of P site-bound tRNA to the platform of the 30S ribosomal subunit and provided additional information about contacts between protein S7 and the anticodon loop in the cleft between the platform and the subunit head.
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PMID:Photoaffinity labeling of 30S-subunit proteins S7 and S11 by 4-thiouridine-substituted tRNA(Phe) situated at the P site of Escherichia coli ribosomes. 929 1

The minimal substrate for human RNase P consists of the 5' leader sequence, aminoacyl acceptor stem, T-stem and T-loop of tRNA. The sequences corresponding to the D-stem, anticodon stem and loop and variable loop are replaced by a bulge which can be as small as 1 nt, but requires > 4 nt for optimal cleavage by RNase P. We found that a trans construct in which the T loop is opened between G57 and A58 (tRNA numbering system) is still processed by RNase P. The strand that is cleaved can be considered the target RNA while the other strand serves as an External Guide Sequence (EGS). We were also able to delete the nucleotides corresponding to nt 58 to 60 in the T-loop without affecting cleavage of the substrate. We propose that the sequence UUCG or UUCA (nucleotide 55 to 57 in the T-loop) positioned 3' to a double helical region of 12 to 13 basepairs containing a bulge of > 4 nt can form a structure that is recognized by human RNase P. The four nucleotides UUCR probably form a structure that resembles the uridine turn in the Tloop of tRNA. Since recognition by RNase P seems to be independent of the helical sequence, we suggest that this motif can be used for targeting RNA molecules for EGS-directed cleavage by RNase P. Based on these results, several 13-mer EGSs targeted to the 2.1 Kb surface antigen mRNA of hepatitis B virus (HBV) were designed and tested using a co-transcriptional cleavage assay with a 2.1 Kb HBV transcript. Some of these were capable of inducing cleavage of the HBV RNA by RNase P. The use of such small EGSs for the inactivation of various genes will be discussed.
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PMID:Design of short external guide sequences (EGSs) for cleavage of target molecules with RNase P. 947 94

In eukaryotes, ribonuclease P (RNase P) requires both RNA and protein components for catalytic activity. The eukaryotic RNase P RNA, unlike its bacterial counterparts, does not possess intrinsic catalytic activity in the absence of holoenzyme protein components. We have used a sensitive photoreactive cross-linking assay to explore the substrate-binding environment for different eukaryotic RNase P holoenzymes. Protein components from the Tetrahymena thermophila and human RNase P holoenzymes form specific products in photoreactions containing [4-thio]-uridine-labeled pre-tRNAGln. The HeLa RNase P RNA in neither the presence nor the absence of holoenzyme protein components formed cross-link products to the pre-tRNAGln probe. Parallel photo-cross-linking experiments with the Escherichia coli RNase P holoenzyme revealed that only the bacterial RNase P RNA forms specific substrate photoadducts. A protein-rich active site for the eukaryotic RNase P represents one unique feature that distinguishes holoenzyme organization between bacteria and eukaryotes.
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PMID:Protein components contribute to active site architecture for eukaryotic ribonuclease P. 951 9

Human RNase P recognizes a small model substrate consisting of only the 5' leader sequence, aminoacyl acceptor stem, and T stem and loop of a tRNA precursor. It was demonstrated here that a bimolecular construct in which the T loop is opened between G57 and A58 (tRNA numbering system) is still processed by RNase P. The strand that is cleaved can be considered the target RNA, whereas the other strand serves as an external guide sequence (EGS). The nucleotides corresponding to nt 58-60 in the T loop could be deleted without affecting cleavage of the substrate. Thus, the complete T loop can be replaced by the single-stranded sequence UUCG or UUCA (nt 55-57 in the T loop). The four nucleotides UUCR possibly form a structure that resembles the uridine turn in the T loop of tRNA. Because recognition by RNase P is independent of the helical sequence, this motif can be used for targeting RNA molecules for EGS-directed cleavage by human RNase P. Chemically modified EGSs with 2'-O-methyl groups also showed activity in inducing RNase P cleavage. Several 13-mer EGSs targeted to the 2.1-kb surface antigen mRNA of hepatitis B virus (HBV) were designed and tested using a co-transcriptional cleavage assay with a 2.1-kb HBV transcript. Some of the new EGSs were capable of inducing cleavage of the HBV RNA by RNase P.
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PMID:Short oligonucleotides as external guide sequences for site-specific cleavage of RNA molecules with human RNase P. 967 Oct 57

The ribonuclease P (RNase P) ribozyme is an endonuclease that binds precursor tRNAs and catalyzes the removal of 5' leader nucleotides. Biochemical and photo-cross-linking studies have identified sites of contact between the mature tRNA domain of pre-tRNA and the ribozyme; however, relatively little is known about the location of the 5' leader in the ribozyme-substrate complex. To investigate the local three-dimensional environment of the 5' leader, we employed the short-range photo-cross-linking agent 4-thiouridine (s(4)U). The s(4)U photoagent was incorporated into a series of pre-tRNA substrates containing unique uridine residues in the 5' leader sequence at positions -1, -3, -5, -7, or -10. The modified substrates formed high-affinity complexes with the ribozyme and produced discrete intermolecular cross-links to RNase P RNA from Bacillus subtilis. Locations of the cross-linked nucleotides in the ribozyme and pre-tRNA were determined by reverse transcriptase primer extension. Photoagents incorporated into the 5' leader detected discrete elements of ribozyme structure in a progression from J18/2 to L15 to P3. Importantly, all of the cross-linked species retained the ability to cleave the covalently attached pre-tRNA, indicating that the cross-links reflect the native structure of the ribozyme-substrate complex. Together with available structural and biochemical data, the cross-linking results suggest a model for the position of the 5' leader within the ground-state ribozyme-substrate complex.
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PMID:The track of the pre-tRNA 5' leader in the ribonuclease P ribozyme-substrate complex. 1050 32

RNase mitochondrial RNA processing (MRP) is a ribonucleoprotein endoribonuclease that is involved in RNA processing events in both the nucleus and the mitochondria. The MRP RNA is both structurally and evolutionarily related to RNase P, the ribonucleoprotein endoribonuclease that processes the 5'-end of tRNAs. Previous analysis of the RNase MRP RNA by phylogenetic analysis and chemical modification has revealed strikingly conserved secondary structural elements in all characterized RNase MRP RNAs. Utilizing successive constraint modeling and energy minimization I derived a three-dimensional model of the yeast RNase MRP RNA. The final model predicts several notable features. First, the enzyme appears to contain two separate structural domains, one that is highly conserved among all MRP and P RNAs and a second that is only conserved in MRP RNAs. Second, nearly all of the highly conserved nucleotides cluster in the first domain around a long-range interaction (LRI-I). This LRI-I is characterized by a ubiquitous uridine base, which points into a cleft between these two structural domains generating a potential active site for RNA cleavage. Third, helices III and IV (the yeast equivalent of the To-binding site) model as a long extended helix. This region is believed to be the binding site of shared proteins between RNase P and RNase MRP and would provide a necessary platform for binding these seven proteins. Indeed, several residues conserved between the yeast MRP and P RNAs cluster in the central region of these helixes. Lastly, characterized mutations in the MRP RNA localize in the model based on their severity. Those mutations with little or no effect on the activity of the enzyme localize to the periphery of the model, while the most severe mutations localize to the central portion of the molecule where they would be predicted to cause large structural defects. Press.
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PMID:Molecular modeling of the three-dimensional architecture of the RNA component of yeast RNase MRP. 1052 8

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)3+(6)) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)3+(6) is a useful spectroscopic probe for Mg(H2O)2+(6)-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)3+(6). We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C.G base pair is replaced by U.G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3' direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)3+(6) and RNA protons were used to locate the site of Co(NH3)3+(6) binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5' by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.
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PMID:Solution structure and metal-ion binding of the P4 element from bacterial RNase P RNA. 1099 99

The solution structures of two 27 nt RNA hairpins and their complexes with cobalt(III)-hexammine [Co(NH(3))(6)(3+)] were determined by NMR spectroscopy. The RNA hairpins are variants of the P4 region from Escherichia coli RNase P RNA: a U-to-A mutant changing the identity of the bulged nucleotide, and a U-to-C, C-to-U double mutant changing only the bulge position. Structures calculated from NMR constraints show that the RNA hairpins adopt different conformations. In the U-to-C, C-to-U double mutant, the conserved bulged uridine in the P4 wild-type stem is found to be shifted in the 3'-direction by one nucleotide when compared with the wild-type structure. Co(NH(3))(6)(3+) is used as a spectroscopic probe for Mg(H(2)O)(6)(2+) binding sites because both complexes have octahedral symmetry and have similar radii. Intermolecular NOE crosspeaks between Co(NH(3))(6)(3+) and RNA protons were used to locate the site of Co(NH(3))(6)(3+) binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop in both mutants, but is shifted 3' by about one base pair in the double mutant. The change of the metal ion binding site is compared with results obtained on corresponding mutant RNase P RNA molecules as reported by Harris and co-workers (RNA, 1, 210-218).
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PMID:Change of RNase P RNA function by single base mutation correlates with perturbation of metal ion binding in P4 as determined by NMR spectroscopy. 1557 80


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