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)

DNA sequences affecting the transcription of the Escherichia coli rnpB transcript encoding the catalytic M1 RNA subunit of RNase P have been analyzed. Previous work (Motamedi, H., Lee, Y., and Schmidt, F.J.) (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3959-3963) identified S1 nuclease protection products corresponding to transcripts originating upstream of the M1 RNA gene. Sequence analysis of the upstream region of rnpB identified three regions homologous to the E. coli consensus promoter sequence. In the present work, analysis of in vitro transcription products by S1 nuclease mapping indicated that all three promoter homologies were capable of directing transcription. The nearest promoter, P-1, was approximately 100 times more active than either of the upstream homologies P-2 and vivo experiments, wherein the three promoter homologies preceding rnpB were cloned into the galactokinase (GalK) expression vector pKO100. The promoter homology nearest to the M1 RNA gene directed the synthesis of GalK above background. The upstream promoter homologies did not direct the synthesis of GalK at a level greater than 1% of transcription from P-1. Deletion of the upstream homologies did not affect transcription from P-1. It was concluded that P-1 is responsible for essentially all M1 RNA transcription in vivo. Single-round transcription experiments in vitro detected strong NusA-independent transcriptional pausing at nucleotides +118 and +121 of the rnpB transcript, with a half-life of 27 s when concentrations of NTPs were near the average Km for elongation. Pausing at these points was eliminated by substitution of ITP for GTP in the transcription mixture. This suggests that pausing is dependent on transcript secondary structure. The position of pausing corresponds to that of a dual stem and loop structure of M1 RNA which has recently been proposed on the basis of phylogenetic sequence analysis.
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PMID:Sites of initiation and pausing in the Escherichia coli rnpB (M1 RNA) transcript. 246 43

In vitro selection techniques are useful means of dissecting the functions of both natural and artificial ribozymes. Using a self-cleaving conjugate containing the Escherichia coli ribonuclease P RNA and its substrate, pre-tRNA (Frank DN, Harris ME, Pace NR, 1994, Biochemistry 33:10800-10808), we have devised a method to select for catalytically active variants of the RNase P ribozyme. A selection experiment was performed to probe the structural and sequence constraints that operate on a highly conserved region of RNase P: the J3/4-P4-J2/4 region, which lies within the core of RNase P and is thought to bind catalytically essential magnesium ions (Harris ME et al., 1994, EMBO J 13:3953-3963; Hardt WD et al., 1995, EMBO J 14:2935-2944; Harris ME, Pace NR, 1995, RNA 1:210-218). We sought to determine which, if any, of the nearly invariant nucleotides within J3/4-P4-J2/4 are required for ribozyme-mediated catalysis. Twenty-two residues in the J3/4-P4-J2/4 component of RNase P RNA were randomized and, surprisingly, after only 10 generations, each of the randomized positions returned to the wild-type sequence. This indicates that every position in J3/4-P4-J2/4 contributes to optimal catalytic activity. These results contrast sharply with selections involving other large ribozymes, which evolve improved catalytic function readily in vitro (Chapman KB, Szostak JW, 1994, Curr Opin Struct Biol 4:618-622; Joyce GF, 1994, Curr Opin Struct Biol 4:331-336; Kumar PKR, Ellington AE, 1995, FASEB J 9:1183-1195). The phylogenetic conservation of J3/4-P4-J2/4, coupled with the results reported here, suggests that the contribution of this structure to RNA-mediated catalysis was optimized very early in evolution, before the last common ancestor of all life.
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PMID:In vitro selection of RNase P RNA reveals optimized catalytic activity in a highly conserved structural domain. 897 68