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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The secondary structure of the 9S RNA precursor to ribosomal 5S RNA in Escherichia coli has been determined using chemical reagents and ribonucleases in combination with a reverse transcription procedure. The 9S RNA precursor was generated in vitro by T7 RNA polymerase, and the rrnB operon terminator, T1, was able to terminate the in vitro transcript. The secondary structure model exhibits three structural domains corresponding to a 5' region, a mature region and a terminator region. The mature domain is structurally identical to 5S RNA, and the ribosomal proteins L18 and L25 are able to bind to the precursor. The processing endoribonuclease RNase E cleaves between the structural domains. Moreover, an intramolecular refolding of the nascent transcript must take place if the current view of RNase III processing stems is correct.
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PMID:The 9S RNA precursor of Escherichia coli 5S RNA has three structural domains: implications for processing. 304 57

To test the ability of an RNA processing enzyme to cleave chemically-modified RNA substrates, RNA transcripts containing RNase III cleavage sites were enzymatically synthesized in vitro to contain specific phosphorothioate diester internucleotide linkages. One transcript (R1.1 RNA) was generated using phage T7 RNA polymerase and a cloned segment of phage T7 DNA containing the R1.1 RNase III processing site. The second transcript was the phage T7 polycistronic early mRNA precursor, which was synthesized using E. coli RNA polymerase and T7 genomic DNA. The RNA transcripts contained phosphorothioate diester groups at positions including the scissile bonds. The modified RNAs were stable to incubation in Mg2+-containing buffer, and were specifically cleaved by RNase III. RNA oligonucleotide sequence analysis showed that the modified R1.1 RNA processing site was the same as the canonical site and contained a phosphorothioate bond. Furthermore, RNase III cleaved the phosphorothioate internucleotide bond with 5' polarity. RNase III cleavage of phosphorothioate substituted T7 polycistronic early mRNA precursor produced the same gel electrophoretic pattern as that obtained with the control transcript. Thus, RNase III cleavage specificity is not altered by phosphorothioate internucleotide linkages.
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PMID:Accurate in vitro cleavage by RNase III of phosphorothioate-substituted RNA processing signals in bacteriophage T7 early mRNA. 327 95

The transcripts covering pnp, the gene encoding polynucleotide phosphorylase, are processed by ribonuclease III. In this study, it is shown that the steady state level of the pnp mRNA increased 11-fold in a ribonuclease III-deficient strain. The synthesis rate of this messenger is only slightly affected in the mutant strain whereas the half-life, which is 1.5 min in the wild type, is considerably increased to more than 40 min. Moreover, polynucleotide phosphorylase is 10-fold over-expressed in the mutant strain, which shows that unprocessed pnp mRNA is functional. The position of the ribonuclease III-sensitive site suggests that the sequence involved in the stabilization of the pnp mRNA is located at the 5' end of the message and that the RNase III processing triggers the decay of the transcripts downstream. A similar function for ribonuclease III in the processing of the messenger for the beta beta' subunits of RNA polymerase is proposed.
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PMID:The first step in the functional inactivation of the Escherichia coli polynucleotide phosphorylase messenger is a ribonuclease III processing at the 5' end. 330 54

Plasmid vectors are described that allow cloning of target DNAs at sites where they will be minimally transcribed by Escherichia coli RNA polymerase but selectively and actively transcribed by T7 RNA polymerase, in vitro or in E. coli cells. Transcription is controlled by the strong phi 10 promoter for T7 RNA polymerase, and in some cases by the T phi transcription terminator. The RNA produced can have as few as two foreign nucleotides ahead of the target sequence or can be cut by RNase III at the end of the target sequence. Target mRNAs can be translated from their own start signals or can be placed under control of start signals for the major capsid protein of T7, with the target coding sequence fused at the start codon or after the 2nd, 11th or 260th codon for the T7 protein. The controlling elements are contained on small DNA fragments that can easily be removed and used to create new expression vectors.
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PMID:Vectors for selective expression of cloned DNAs by T7 RNA polymerase. 331 56

A gene expression system based on bacteriophage T7 RNA polymerase has been developed. T7 RNA polymerase is highly selective for its own promoters, which do not occur naturally in Escherichia coli. A relatively small amount of T7 RNA polymerase provided from a cloned copy of T7 gene 1 is sufficient to direct high-level transcription from a T7 promoter in a multicopy plasmid. Such transcription can proceed several times around the plasmid without terminating, and can be so active that transcription by E. coli RNA polymerase is greatly decreased. When a cleavage site for RNase III is introduced, discrete RNAs of plasmid length can accumulate. The natural transcription terminator from T7 DNA also works effectively in the plasmid. Both the rate of synthesis and the accumulation of RNA directed by T7 RNA polymerase can reach levels comparable with those for ribosomal RNAs in a normal cell. These high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and/or stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degradation. It seems likely that a specific mRNA produced by T7 RNA polymerase can rapidly saturate the translational machinery of E. coli, so that the rate of protein synthesis from such an mRNA will depend primarily on the efficiency of its translation. When the mRNA is efficiently translated, a target protein can accumulate to greater than 50% of the total cell protein in three hours or less. We have used two ways to deliver active T7 RNA polymerase to the cell; infection by a lambda derivative that carries gene 1, or induction of a chromosomal copy of gene 1 under control of the lacUV5 promoter. When gene 1 is delivered by infection, very toxic target genes can be maintained silent in the cell until T7 RNA polymerase is introduced, when they rapidly become expressed at high levels. When gene 1 is resident in the chromosome, even the very low basal levels of T7 RNA polymerase present in the uninduced cell can prevent the establishment of plasmids carrying toxic target genes, or make the plasmid unstable.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. 353 5

The RNA polymerases encoded by bacteriophages T3 and T7 have similar structures, but exhibit nearly exclusive template specificities. We have determined the nucleotide sequence of the region of T3 DNA that encodes the T3 RNA polymerase (the gene 1.0 region), and have compared this sequence with the corresponding region of T7 DNA. The predicted amino acid sequence of the T3 RNA polymerase exhibits very few changes when compared to the T7 enzyme (82% of the residues are identical). Significant differences appear to cluster in three distinct regions in the amino-terminal half of the protein. Analysis of the data from both enzymes suggests features that may be important for polymerase function. In particular, a region that differs between the T3 and T7 enzymes exhibits significant homology to the bi-helical domain that is common to many sequence-specific DNA binding proteins. The region that flanks the structural gene contains a number of regulatory elements including: a promoter for the E. coli RNA polymerase, a potential processing site for RNase III and a promoter for the T3 polymerase. The promoter for the T3 RNA polymerase is located only 12 base pairs distal to the stop codon for the structural gene.
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PMID:Sequence and analysis of the gene for bacteriophage T3 RNA polymerase. 390 58

rRNA genes of Caulobacter crescentus CB13 were isolated and shown to be present in two gene clusters in the genome. The organization of each rRNA gene cluster was found to be 5'-16S-tRNA spacer-23S-5S-3'. The DNA sequence of 40% of the 16S rRNA gene, the entire 16S/23S intergenic spacer region, and portions of the 23S rRNA gene were determined. Analysis of the nucleotide sequence in the 16S-23S intergenic spacer region revealed the presence of tRNAIle and tRNAAla genes. Large invert repeat sequences were found surrounding the 16S rRNA gene. These inverted repeat sequences are analogous to the RNase III-processing sites in the E. coli rRNA precursor. Small invert repeat sequences were also found flanking the individual tRNA genes. RNA polymerase-binding studies with restriction fragments of the rRNA gene cluster revealed three regions which bound enzyme, and these regions were shown to contain transcription initiation sites. One of these sites was located within the 16S gene near its 3' end, and the other two were found at the 5' end of the 23S gene.
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PMID:Organization and nucleotide sequence analysis of an rRNA and tRNA gene cluster from Caulobacter crescentus. 400 39

Transcription of T7 DNA by purified Escherichia coli RNA polymerase without added factors produces long RNA molecules that begin near the left end of T7 DNA and terminate at the end of the early region. An endonuclease has been isolated from uninfected E. coli that cleaves these long RNAs at five specific sites to generate RNA molecules essentially the same as the early T7 RNAs observed in vivo. This sizing factor, which may be RNase III, can act during or after RNA synthesis. Synthesis of early RNA chains has been shown to start at three strong initiators, spaced about 150-200 base-pairs apart near the left end of T7 DNA. Thus, the five cleavages by sizing factor generate the five early messenger RNAs of T7 plus three overlapping RNAs from the promoter region. RNA chains that are started at two of the strong initiators begin with A; those started at the third begin with G. A few minor initiators have also been observed, from which only short chains seem to be synthesized. Their locations in T7 DNA have not been mapped. Rho factor does not appear to be needed to generate any of these early T7 RNAs.
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PMID:T7 early RNAs are generated by site-specific cleavages. 457 24

The early region of T7 DNA is transcribed as a single unit in a Ribonuclease III-deficient E. coli strain to produce large molecules essentially identical to those produced in vitro by E. coli RNA polymerase. As with the in vitro RNAs, these molecules are cut by purified RNase III in vitro to produce the messenger RNAs normally observed in vivo. Thus, the normal pathway for producing the T7 early messenger RNAs in vivo appears to involve endonucleolytic cleavage by RNase III. The uninfected RNase III-deficient strain contains several RNAs not observed in the parent strain. Patterns of labeling in vivo suggest that the largest of these RNAs, about 1.8 x 10(6) daltons, may be a precursor to the 16S and 23S ribosomal RNAs. When this large molecule is treated in vitro with purified RNase III, molecules the size of precursor 16S and 23S ribosomal RNAs are released; hybridization competition experiments also indicate that the 1.8 x 10(6) dalton RNA does indeed represent ribosomal RNA. Thus, RNase III cleavage seems to be part of the normal pathway for producing at least the 16S and 23S ribosomal RNAs in vivo. Several smaller molecules are also released from the 1.8 x 10(6) dalton RNA by RNase III, but it is not yet established whether any of these contain 5S RNA sequences.
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PMID:T7 early RNAs and Escherichia coli ribosomal RNAs are cut from large precursor RNAs in vivo by ribonuclease 3. 458 48

Attenuation and processing of the mRNA from the ribosomal protein-RNA polymerase operon rplJL--rpoBC have been demonstrated by the analysis of nuclease SI-resistant RNA . DNA hybrids. These hybrids were formed between RNA produced in vivo and specific DNA restriction fragments which span the rplL--rpoB intercistronic region. The 3' end of the predominant attenuated RNA lies 69 nucleotides beyond the end of the rplL gene following sequence features that are similar to those of other known attenuators. The nonattenuated transcript is normally cleaved in the intercistronic region. However, in an RNase III mutant strain, the hybrids corresponding to the cleaved nonattenuated mRNAs disappear and the expected full-sized hybrid is seen. We have localized the cleavage to an area of possible secondary structure in the transcript approximately 200 nucleotides beyond the end of the rplL gene. This demontrates RNase III processing of Escherichia coli mRNA. The methods used in this study permit the examination of specific ends of large and complex polycistronic mRNAs. Such experiments should help in understanding how posttranscriptional events influence gene expression.
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PMID:Attenuation and processing of RNA from the rplJL--rpoBC transcription unit of Escherichia coli. 615 44


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