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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcription initiation has been shown to occur in vitro at several sites within a cloned Caulobacter crescentus ribosomal RNA gene cluster that lacks the major promoter region 5' to the 16 S rRNA gene. The predominant transcription start site in vitro was located near the 3' end of the 16 S rRNA gene. Transcription initiation from this region was also detected in vivo, when the cloned rRNA gene cluster was present on a multi-copy plasmid. The transcription start sites in vitro and in vivo were shown to be identical by S1 nuclease mapping and were found to be located approximately 300 nucleotides upstream from the 3' end of the 16 S rRNA gene. The transcript synthesized in vitro was shown to be cleaved by C. crescentus RNase III and to release the transfer RNA genes from the downstream 16 S/23 S intergenic spacer region. Analysis of the nucleotide sequence near the internal 16 S rRNA transcription start site revealed the presence of a consensus promoter sequence followed by the beginning of an open reading frame approximately 90 nucleotides downstream. Examination of the 16 S rRNA genes from other bacterial species and chloroplasts and 18 S rRNA genes from Xenopus and yeast revealed that the nucleotide sequence of this internal 16 S rRNA promoter region was highly conserved. Although the length of these 16 S and 18 S rRNA genes is slightly variable, the distance of the conserved promoter sequence from the 3' end of these genes has been conserved.
J Mol Biol 1986 Jan 05
PMID:Transcription initiation in vitro and in vivo at a highly conserved promoter within a 16 S ribosomal RNA gene. 242 Sep 95

Transcripts from the rplKAJL-rpoBC ribosomal protein-RNA polymerase gene cluster have been quantified and their ends mapped using RNA-DNA hybridization, sucrose density-gradient sedimentation, Northern hybridization and S1 nuclease protection. The results indicate that the most abundant transcript is the 2600 nucleotide tetracistronic L11-L1-L10-L12 mRNA initiated at the upstream major PL11 promoter and terminated at the transcription attenuator in the L12-beta intergenic space. Somewhat less abundant 1300 nucleotide L11-L1 and L10-L12 bicistronic transcripts were observed. The 3' ends of the L11-L1 transcripts were heterogeneous; most of the ends were localized to three sites within a 110 base-pair region in the L1-L10 intergenic space. This intergenic space encodes also the major PL10 promoter and the mRNA binding site for the L10 translational control protein. Two 5' ends were observed for L10-L12 bicistronic mRNA, one at the PL10 promoter and the other 150 nucleotides further downstream in a region in which promoter activity has not been detected. It is suggested that this second downstream 5' end is generated by processing of the transcripts initiated at the major PL10 promoter. No transcript initiation in the L10-L12 intergenic space was detected. About 80% of the transcripts reading through the L12 gene were terminated in the vicinity of the transcription attenuator that is responsible for the reduction in the expression of the downstream RNA polymerase genes. Transcripts reading through the attenuator were partially processed by RNase III within a potential hairpin structure in the RNA transcript. Processing appears to produce 3' and 5' transcript end sites separated by about ten nucleotides. No other major 5' ends were observed in the L12-beta intergenic space. These results indicate that the two major promoters, PL11 and PL10, are both utilized to drive the interrelated transcriptional expression of this ribosomal protein-RNA polymerase gene cluster.
J Mol Biol 1987 Apr 20
PMID:Transcription products from the rplKAJL-rpoBC gene cluster. 244 6

The metY gene coding for a minor form of the initiator tRNA is the first gene of a complex polycistronic operon also encoding the transcription termination factor NusA and the translation initiation factor IF2. The mixed tRNA-mRNA polycistronic transcript is cleaved by RNase III in a hairpin structure downstream from the tRNA. This cleavage separates the tRNA from the mRNA and initiates the rapid degradation of the 5' extremity of the downstream mRNA. Dissociation of the structural (tRNA) and informational (mRNA) RNAs from this operon is also achieved by independent transcription in vivo. The presence of two transcription terminators located downstream from metY produces a small tRNAMetf2 precursor transcript, whereas an internal promoter situated between metY and the first open reading frame directs the transcription of only the protein-coding part of the operon.
J Mol Biol 1989 Nov 20
PMID:Cleavage by RNase III in the transcripts of the met Y-nus-A-infB operon of Escherichia coli releases the tRNA and initiates the decay of the downstream mRNA. 248 Oct 42

We have isolated on a multicopy plasmid a mutant rrnB ribosomal RNA operon containing a 130 base-pair deletion immediately preceding the 23 S rRNA gene. The deletion shortens by just three base-pairs the 26 base-pair complementarity of the sequences that flank the 23 S rRNA gene, and which normally form an RNase III cleavage site in the rrnB primary transcript. Both in vivo and in vitro, cleavage at the altered RNase III site was almost completely abolished by the mutation. Our results therefore indicate that even a small perturbation of the double-stranded region normally recognized by RNase III strongly inhibits the action of the enzyme.
J Mol Biol 1985 Mar 20
PMID:A mutation in an Escherichia coli ribosomal RNA operon that blocks the production of precursor 23 S ribosomal RNA by RNase III in vivo and in vitro. 258 39

The synthesis of Escherichia coli polynucleotide phosphorylase (PNPase) was examined in a mutant strain defective in the RNA processing enzyme RNase III (Rnc-). We found that the specific activity and the synthesis rate of PNPase were increased in the Rnc- strain by more than three times that in an Rnc+ strain. Such increased synthesis of PNPase was not observed in a mutant strain transformed with a plasmid carrying the rnc+ gene. Quantitative analysis of RNA showed that the transcripts from the pnp gene, which encodes PNPase, were degraded more slowly in the Rnc- strain than in the Rnc+ strain. These results indicate that processing of the transcripts by RNase III is intimately involved in controlling the expression of pnp by affecting the stability of its messenger RNA.
Mol Gen Genet 1987 Aug
PMID:RNA processing by RNase III is involved in the synthesis of Escherichia coli polynucleotide phosphorylase. 282 71

Bacteriophage lambda int gene expression is regulated differentially from transcripts originated at the pL and pI promoters. Transcripts initiated at pI terminate at the site tI and express int gene product efficiently. Polymerases starting at pL do not terminate at tI, due to the antiterminating activity of lambda N protein. The pL transcripts are unable to express Int protein efficiently because sib, a control site overlapping tI in the unterminated RNA, is processed by host RNase III. We have isolated lambda sib- mutants by their inability to inhibit int expression from pL transcripts. sib mutations were genetically mapped to the left of the lambda attachment site, and do not structurally alter this site for recombination. Several sib mutations do alter the nucleotide sequence of the overlapping sib and tI sites. The lambda sib- mutants tested prevent RNA processing but do not affect transcription termination in vivo.
J Mol Biol 1986 Sep 05
PMID:Mutations of bacteriophage lambda that define independent but overlapping RNA processing and transcription termination sites. 294 21

The accessibility of ds- and ss-segments of phage MS2 RNA to ds- and ss-specific nucleases (RNase III, nuclease SV and nuclease S1) was studied. The results show that the RNA has hydrolysis sites for all the nucleases used. These sites are unvariable in a wide range of the conditions (ionic strength, pH, bivalent cations and temperature) and are not changed also after denaturation-renaturation of the RNA. This testifies that the distribution and interactions of ds- and ss-segments in the whole molecule are very specific and stable.
Mol Biol Rep 1985 Apr
PMID:The accessibility of phage MS2 RNA to structure specific nucleases in various conditions. 299 49

The rpsO gene of Escherichia coli, which encodes ribosomal protein S15 is located at 69 minutes on the chromosome. It is adjacent to the pnp gene, which encodes polynucleotide phosphorylase. The two genes are separated by 249 nucleotides and are transcribed in the same direction. We report here in vivo S1 nuclease mapping and in vitro transcription experiments that demonstrate that rpsO and pnp are cotranscribed from a promoter P1, located 108 nucleotides upstream from rpsO, and that another promoter P2, located between the two genes 158 nucleotides upstream from pnp, also directs the transcription of pnp. Transcription from P1 can either terminate at the terminator t1 identified in vivo and in vitro, 18 nucleotides downstream from rpsO, or transcribe through t1 and into pnp. Comparison of the transcripts synthesized in wild-type and RNase III-deficient strains of E. coli shows that all the P1 readthrough transcripts and P2 transcripts are cleaved by RNase III. Two specific cuts are made by RNase III in a double-stranded structure about 100 nucleotides upstream rpsO. We also found that some transcripts of this operon start 47 nucleotides downstream from rpsO, in the region of t1. No promoter has been identified in this region. This mRNA is attributed to an endonucleolytic cleavage of the polycistronic transcripts and the location of the cut is named M. The order of the transcription signals and of the maturation sites in relation to rpsO and pnp can be summarized as follows: P1, rpsO, t1, M, P2, RNase III-processing sites, pnp. The possible roles of mRNA processing events in the expression of rpsO-pnp operon are discussed.
J Mol Biol 1986 Jan 05
PMID:Initiation, attenuation and RNase III processing of transcripts from the Escherichia coli operon encoding ribosomal protein S15 and polynucleotide phosphorylase. 300 65

S1 nuclease mapping was performed on transcripts from the major leftward operon of the bacteriophage lambda in order to locate the 3' ends of stable RNA species produced in vivo. The analysis was carried out on RNA purified from either an induced lambda prophage or bacteria carrying a plasmid containing a large segment of lambda including the intact PL operon through the bet gene. The S1 nuclease mapping was performed on transcripts produced in the presence and the absence of the N antitermination function, and in the presence and the absence of either the RNase III processing enzyme or the Rho factor. The results of this work indicate that the intercistronic region between the N and ral genes of lambda contains three sites at which transcripts end under N-Rho+ conditions (positions on the lambda sequence: 34,826, 34,558 and 34,393). The distal two correspond to the two sites previously described in this region as tL1 (on both sides of the BamHI site). In the region between ral and Ea10, we mapped the 3' ends of three species of RNA. The 3' end of one species was found to be located 90 nucleotides proximal to tL2a, at 34,000 in the lambda sequence. The terminator at this site may be partially N-resistant. In an RNase III deficient host, an additional RNA species is formed. The 3' end of this RNA species is located at tL2a (33,910 on the lambda sequence). In the presence of the antitermination N gene product, the readthrough transcripts are processed to form a 3' end at position 33,980 on the lambda sequence. These results suggest that elongation of transcription of the lambda PL operon is reduced gradually by clusters of termination located between genes and that the expression of the terminated products is further controlled by processing of the mRNA.
J Mol Biol 1986 May 05
PMID:Transcription termination and processing sites in the bacteriophage lambda pL operon. 302 19

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)
J Mol Biol 1986 May 05
PMID:Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. 353 5


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