Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Though up to 20% of the total RNA in bacterial cells is tRNA, the regulation of tRNA distribution on the genomic level remains unclear. tRNA distribution is governed by four processes: transcription, processing of precursor tRNA, degradation of precursor tRNA and degradation of mature tRNA. To elucidate the relationship between these processes in the regulation of tRNA production, the relative tRNA distribution was measured using a microarray specifically designed for tRNA. We developed a procedure that selectively labels 3'-CCA-containing RNAs with the fluorophores Cy3 or Cy5. The labeled tRNAs were then hybridized to microarrays printed with complementary DNA probes. The regulation of tRNA distribution in Bacillus subtilis was explored for a wild-type strain and a mutant strain with significantly decreased levels of RNase P, the enzyme required for the 5' maturation of all tRNA. The strains were either grown under a variety of conditions at doubling times ranging from 0.1 to 2.2 doublings per hour to investigate growth-related changes in the tRNA abundance or treated with the transcriptional inhibitor rifampicin to analyze mature tRNA degradation. Our results confirm that transcription and processing contribute significantly to the distribution of the 35 tRNA species in B.subtilis, and suggest a role for the degradation of precursor tRNA. Mature tRNA degradation occurs with little specificity for individual tRNA species and on the hour time-scale, indicating that degradation of mature tRNA plays only a minor role in the regulation of tRNA distribution. Aside from transcription, the final tRNA distribution appears to be derived from a balance between processing and precursor degradation activities.
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PMID:Exploring the regulation of tRNA distribution on the genomic scale. 1500 50

It has been postulated that a highly reduced form of transfer messenger RNA (tmRNA), a bacterial molecule involved in the rescue of stalled ribosomes during translation, is expressed in the mitochondrion of the jakobid Reclinomonas americana. Here we show that genes encoding both one-piece and two-piece tmRNAs are present in six different jakobid mitochondrial DNAs. Mitochondrial tmRNAs have retained the highly conserved tRNA(Ala)-like domain, but they apparently lack the mRNA-like region present in all bacterial tmRNAs. Comparative analysis of jakobid mitochondrial genomes shows that a potential mRNA-like region in R. americana (orf64) is located at distant genomic positions in other jakobids. Our results strongly suggest that orf64 is a tatA homolog. Through Northern hybridization we confirm the postulated reduced size of both a one-piece tmRNA in Jakoba libera and a two-piece tmRNA in Seculamonas ecuadoriensis. The J. libera tmRNA is post-transcriptionally modified by addition of a 3' CCA tail, processed in vitro by RNase P RNA, and specifically charged with alanine in vitro by alanyl-tRNA synthetase. Our results strongly support the functionality of these reduced mitochondrial tmRNAs.
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PMID:Loss of the mRNA-like region in mitochondrial tmRNAs of jakobids. 1503 63

Transfer RNAs (tRNAs) are synthesized as part of longer primary transcripts that require processing of both their 3' and 5' extremities in every living organism known. The 5' side is processed (matured) by the ubiquitously conserved endonucleolytic ribozyme, RNase P, whereas removal of the 3' tails can be either exonucleolytic or endonucleolytic. The endonucleolytic pathway is catalysed by an enzyme known as RNase Z, or 3' tRNase. RNase Z cleaves precursor tRNAs immediately after the discriminator base (the unpaired nucleotide 3' to the last base pair of the acceptor stem, used as an identity determinant by many aminoacyl-tRNA synthetases) in most cases, yielding a tRNA primed for addition of the CCA motif by nucleotidyl transferase. Here we report the crystal structure of Bacillus subtilis RNase Z at 2.1 A resolution, and propose a mechanism for tRNA recognition and cleavage. The structure explains the allosteric properties of the enzyme, and also sheds light on the mechanisms of inhibition by the CCA motif and long 5' extensions. Finally, it highlights the extraordinary adaptability of the metallo-hydrolase domain of the beta-lactamase family for the hydrolysis of covalent bonds.
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PMID:Structural basis for substrate binding, cleavage and allostery in the tRNA maturase RNase Z. 1565 28

Transfer RNAs are transcribed as precursors with extensions at both the 5' and 3' ends. RNase P removes endonucleolytically the 5' end leader. tRNase Z can remove endonucleolytically the 3' end trailer as a necessary step in tRNA maturation. CCA is not transcriptionally encoded in the tRNAs of eukaryotes, archaebacteria and some bacteria and must be added by a CCA-adding enzyme after removal of the 3' end trailer. tRNase Z is a member of the beta-lactamase family of metal-dependent hydrolases, the signature sequence of which, the conserved histidine cluster (HxHxDH), is essential for activity. Starting with baculovirus-expressed fruit fly tRNase Z, we completed an 18 residue Ala scan of the His cluster to analyze the functional landscape of this critical region. Residues in and around the His cluster fall into three categories based on effects of the substitutions on processing efficiency: substitutions in eight residues have little effect, five substitutions reduce efficiency moderately (approximately 5-50-fold), while substitutions in five conserved residues, one serine, three histidine and one aspartate, severely reduce efficiency (approximately 500-5000-fold). Wild-type and mutant dissociation constants (Kd values), determined using gel shifts, displayed no substantial differences, and were of the same order as kM (2-20 nM). Lower processing efficiencies arising from substitutions in the His domain are almost entirely due to reduced kcat values; conserved, functionally important residues within the His cluster of tRNase Z are thus involved in catalysis, and substrate recognition and binding functions must reside elsewhere in the protein.
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PMID:Residues in the conserved His domain of fruit fly tRNase Z that function in catalysis are not involved in substrate recognition or binding. 1593 79

We showed previously that the bacterial ribonuclease P (RNase P) ribozyme has substrate shape preference depending on the concentrations of catalytically important magnesium ions. The ribozyme discriminates a canonical cloverleaf precursor tRNA from a hairpin RNA with a CCA-tag sequence at low concentrations of magnesium ions. By detailed analysis of the shape preference using the bottom-half part-shifting variants of a tRNA precursor, we showed that the RNAs in a T-shape structure can be substrates for the ribozyme reactions even at low concentrations of magnesium ions, and that the RNA in a natural L-shape is the best substrate for both the ribozyme and the holo enzyme. The results also showed that the position of the bottom-half part did not affect the cleavage site selection of a substrate by the enzyme. Our results are the first kinetic evidence to show the importance of the bottom-half part of tRNA molecule, and our result also showed that the holo enzyme can discriminate substrate shape as well as the ribozyme at low concentrations of metal ions.
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PMID:Substrate shape preference of Escherichia coli ribonuclease P ribozyme and holo enzyme using bottom-half part-shifting variants of pre-tRNA. 1624 56

A sequence-specific M1GS ribozyme (M1-T3) was constructed by covalently linking an oligonucleotide (guide sequence,GS) to the 3' terminus of M1 RNA ,the catalytic subunit of RNase P from Escherichia coli. The engineered ribozyme is targeted to the mRNA sequence encoding a protein kinase (UL97) of HCMV and could effectively cleave the mRNA segment in vitro. Further studies about the significance of some structural elements in the M1 GS (e.g. the 3' CCA tail sequence and a bridge sequence between the 3' terminus of M1 RNA and the 5' terminus of the GS) were carried out. The results showed that the bridge sequence of 88 nucleotides in a mutated M1 GS (i.e. M1-T3*) dramatically increased the cleavage activity to the substrate in vitro. Moreover, the 3'CCA tail sequence was confirmed to be a necessary element for the cleavage activity of M1 GS ribozyme. These data we got in the study will help in understanding the interaction between the M1 GS RNA and its substrate,and will markedly facilitate the research of a general gene targeting agent for anti-HCMV applications.
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PMID:Construction of an effective m1 GS ribozyme targeting HCMV UL97 mRNA segment in vitro. 1631 87

The L15 region of Escherichia coli RNase P RNA forms two Watson-Crick base pairs with precursor tRNA 3'-CCA termini (G292-C75 and G293-C74). Here, we analyzed the phenotypes associated with disruption of the G292-C75 or G293-C74 pair in vivo. Mutant RNase P RNA alleles (rnpBC292 and rnpBC293) caused severe growth defects in the E. coli rnpB mutant strain DW2 and abolished growth in the newly constructed mutant strain BW, in which chromosomal rnpB expression strictly depended on the presence of arabinose. An isosteric C293-G74 base pair, but not a C292-G75 pair, fully restored catalytic performance in vivo, as shown for processing of precursor 4.5S RNA. This demonstrates that the base identity of G292, but not G293, contributes to the catalytic process in vivo. Activity assays with mutant RNase P holoenzymes assembled in vivo or in vitro revealed that the C292/293 mutations cause a severe functional defect at low Mg2+ concentrations (2 mM), which we infer to be on the level of catalytically important Mg2+ recruitment. At 4.5 mM Mg2+, activity of mutant relative to the wild-type holoenzyme, was decreased only about twofold, but 13- to 24-fold at 2 mM Mg2+. Moreover, our findings make it unlikely that the C292/293 phenotypes include significant contributions from defects in protein binding, substrate affinity, or RNA degradation. However, native PAGE experiments revealed nonidentical RNA folding equilibria for the wild-type versus mutant RNase P RNAs, in a buffer- and preincubation-dependent manner. Thus, we cannot exclude that altered folding of the mutant RNAs may have also contributed to their in vivo defect.
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PMID:The precursor tRNA 3'-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo. 1713 88

For catalysis by bacterial type B RNase P, the importance of a specific interaction with p(recursor)tRNA 3'-CCA termini is yet unclear. We show that mutation of one of the two G residues assumed to interact with 3'-CCA in type B RNase P RNAs inhibits cell growth, but cell viability is at least partially restored at increased RNase P levels due to RNase P protein overexpression. The in vivo defects of the mutant enzymes correlated with an enzyme defect at low Mg(2+) in vitro. For Bacillus subtilis RNase P, an isosteric C259-G(74) bp fully and a C258-G(75) bp slightly rescued catalytic proficiency, demonstrating Watson-Crick base pairing to tRNA 3'-CCA but also emphasizing the importance of the base identity of the 5'-proximal G residue (G258). We infer the defect of the mutant enzymes to primarily lie in the recruitment of catalytically relevant Mg(2+), with a possible contribution from altered RNA folding. Although with reduced efficiency, B. subtilis RNase P is able to cleave CCA-less ptRNAs in vitro and in vivo. We conclude that the observed in vivo defects upon disruption of the CCA interaction are either due to a global deceleration in ptRNA maturation or severe inhibition of 5'-maturation for a ptRNA subset.
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PMID:In vivo and in vitro investigation of bacterial type B RNase P interaction with tRNA 3'-CCA. 1735 91

Ribonuclease P (RNase P) is involved in the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). We have found that RNase P RNA (PhopRNA) and five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) reconstitute RNase P activity with enzymatic properties similar to those of the authentic ribozyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. We report here that nucleotides A40, A41, and U44 at helix P4, and G269 and G270 located at L15/16 in PhopRNA, are, like the corresponding residues in Esherichia coli RNase P RNA (M1RNA), involved in hydrolysis by coordinating catalytic Mg(2+) ions, and in the recognition of the acceptor end (CCA) of pre-tRNA by base-pairing, respectively. The information reported here strongly suggests that PhopRNA catalyzes the hydrolysis of pre-tRNA in approximately the same manner as eubacterial RNase P RNAs, even though it has no enzymatic activity in the absence of the proteins.
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PMID:Identification of nucleotide residues essential for RNase P activity from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. 1769 Apr 61

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that generates the mature 5' ends of tRNAs. Ubiquitous across all three kingdoms of life, the composition and functional contributions of the RNA and protein components of RNase P differ between the kingdoms. RNA-alone catalytic activity has been reported throughout bacteria, but only for some archaea, and only as trace activity for eukarya. Available information for RNase P from photosynthetic organelles points to large differences to bacterial as well as to eukaryotic RNase P: for spinach chloroplasts, protein-alone activity has been discussed; for RNase P from the cyanelle of the glaucophyte Cyanophora paradoxa, a type of organelle sharing properties of both cyanobacteria and chloroplasts, the proportion of protein was found to be around 80% rather than the usual 10% in bacteria. Furthermore, the latter RNase P was previously found catalytically inactive in the absence of protein under a variety of conditions; however, the RNA could be activated by a cyanobacterial protein, but not by the bacterial RNase P protein from Escherichia coli. Here we demonstrate that, under very high enzyme concentrations, the RNase P RNA from the cyanelle of C. paradoxa displays RNA-alone activity well above the detection level. Moreover, the RNA can be complemented to a functional holoenzyme by the E. coli RNase P protein, further supporting its overall bacterial-like architecture. Mutational analysis and domain swaps revealed that this A,U-rich cyanelle RNase P RNA is globally optimized but conformationally unstable, since changes as little as a single point mutation or a base pair identity switch at positions that are not part of the universally conserved catalytic core led to a complete loss of RNA-alone activity. Likely related to this low robustness, extensive structural changes towards an E. coli-type P5-7/P15-17 subdomain as a canonical interaction site for tRNA 3'-CCA termini could not be coaxed into increased ribozyme activity.
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PMID:RNase P of the Cyanophora paradoxa cyanelle: a plastid ribozyme. 1788 Nov 13


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