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)

Transcription of the Caulobacter crescentus phage phi Cd1 genome requires both the host RNA polymerase and a phage-encoded, rifampicin-resistant RNA polymerase. Transcription of the early region of the phi Cd1 genome was examined in vitro with C. crescentus RNA polymerase. Four transcripts, A, B, C, and D, which ranged in size from 2.9 X 10(6) to 0.53 X 10(6) daltons, were synthesized in vitro by the holoenzyme. Transcript A appeared to be the major transcript since (a) it was the size of the entire 20% of the genome shown in vivo to code for the early phage mRNA, (b) it was one of the first transcripts synthesized at low enzyme-to-DNA molar ratios, and (c) it was synthesized in approximately 3 times the molar equivalent observed for the other transcripts. The A transcript initiated primarily with GTP although a portion was also labeled with ATP. The B, C, and D transcripts were present in equivalent molar ratios, were all smaller than transcript A, and were found to yield RNase III digestion products that were subsets of each other as well as of transcript A. Each of these transcripts proved to be a de novo transcript since (a) each could be pulse labeled during the initial 20 s of the reaction and (b) each transcript contained a triphosphate at its 5' terminus. Evidence is presented that suggests that the B and C transcripts initiate at or near the major A promoter but terminate at different termination or pause sites within the early region of the phage genome. Transcript D appears to initiate at a minor promoter within the terminally redundant region of the genome preceding the A promoter.
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PMID:In vitro transcription of the early region of Caulobacter phage phi Cd1 deoxyribonucleic acid by host RNA polymerase. 629 89

Escherichia coli ribonuclease H was purified to near-homogeneity and identified as the only additional factor required for initiation of in vitro Co1E1 DNA replication from the unique origin by RNA polymerase and DNA polymerase I. Both ribonuclease H activity and stimulating activity for Co1E1 DNA synthesis comigrate with the single protein band in gel electrophoresis. These two activities coincide throughout the process of purification. Some DNA synthesis takes place on covalently closed-circular DNA molecules other than Co1E1 DNA with the three purified enzymes. This DNA synthesis is suppressed by an Escherichia coli single-strand DNA binding protein and/or a high concentration of ribonuclease H. Negative superhelicity of template DNA is required for efficient primer formation. No evidence that supports involvement of ribonuclease III in initiation of Co1E1 DNA replication or its regulation was found.
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PMID:Purification of ribonuclease H as a factor required for initiation of in vitro Co1E1 DNA replication. 629 61

The DNA sequences of three major class III T3 RNA polymerase promoters located at 45.0, 55.0, and 64.8% on the standard T3 genetic map have been determined. The precise RNA initiation sites were also determined by 5'-terminal RNA sequence analysis of the transcripts synthesized from the promoter-containing DNA fragments. Alignment of these three class III promoters and a previously determined T3 RNA polymerase promoter at 1.05% on T3 genetic map, with start points of transcription (+1) in register, indicates a high degree of sequence conservation among the four T3 RNA polymerase promoters. The sequences are identical between positions -12 and +4 and are uniformly A-T between -12 and -17. The conserved portion of the (-) strand sequence is 5' (sequence in text) Upstream from -17 and downstream from +4 the sequences diverge. Comparison of this sequence with a prototype 23-base pair promoter sequence for T7 RNA polymerase shows overall homology between positions -17 and +4 with conserved divergence at residues -2 and between -10 and -12. Furthermore, careful examination of the nucleotide sequences around 45.0 and 64.8 T3 map units shows that the putative RNA sequences arising from these regions by overlapping transcription from upstream promoters can be arranged into stable stemloop structures thought to be required for RNase III cleavage. This pattern is similar to those reported for the corresponding T7 RNA polymerase promoters on T7 DNA (Dunn. J. J., and Studier, F. W. (1983) J. Mol. Biol. 166, 477-535).
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PMID:Locations and nucleotide sequences of three major class III promoters for bacteriophage T3 RNA polymerase on T3 DNA. 631 18

The 319 nucleotide long intergenic region between the rplL (L12) and the rpoB (beta) genes of the L10 operon contains a transcription attenuation sequence and a RNase III mRNA processing sequence. Four site specific deletions located within this intergenic space which remove either the transcription attenuation sequence or the RNase III mRNA processing sequence or both sequences have been isolated on recombinant DNA plasmids carrying this operon. Deletions of sequences surrounding the RNase III processing site result in a uniform 80-90% reduction in the translational efficiency of beta subunit mRNA. This reduction in translation efficiency appears not to be related to processing per se; transcription of the rpoB and rpoC genes and the translation efficiency of the respective mRNA sequences were indistinguishable in an RNase III processing defective mutant (rnc) and its isogenic parent (rnc+). Deletions of the attenuator sequence result in a substantial increase in the downstream transcription of the beta subunit gene. The translational efficiency of RNase III processed beta subunit mRNA was found to be related in an inverse manner to the level of beta subunit synthesis. These result suggest that sequences on the mRNA in the vicinity of the RNase III processing site (i) are essential for efficient translation of beta subunit mRNA and (ii) are utilized for reducing the translational efficiency of the beta subunit mRNA when the beta subunit protein is produced in excess of that required for RNA polymerase assembly.
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PMID:Site specific deletions of regulatory sequences in a ribosomal protein-RNA polymerase operon in Escherichia coli. Effects on beta and beta' gene expression. 632 99

An RNA processing enzyme has been isolated from Caulobacter crescentus which is specific for double-stranded RNA, has an absolute requirement for monovalent cations, and can be eluted from a poly I:C agarose affinity column in pure form. This enzyme, like RNase III isolated from Escherichia coli, processes precursor ribosomal RNAs and polycistronic phage mRNAs and has a monomeric Mr of approximately 20,000. The two enzymes differ, however, in the recognition of specific cleavage sites and yield different digestion products when either coliphage T7 or C. crescentus phage phi Cdl early mRNA is used as substrate. Two lines of evidence are presented which show that an RNase III activity functions as a processing enzyme in C. crescentus. (a) In an in vitro reaction, C. crescentus phage phi Cdl major early mRNA synthesized in vitro by host RNA polymerase was processed by RNase III to yield RNA species which co-migrated with phage RNA synthesized in vivo in phi Cdl-infected cells, and (b) an in vitro transcript of a C. crescentus DNA clone containing the entire 16 S gene and part of the 23 S gene was processed by C. crescentus RNase III to yield an RNA product which co-migrated with 16 S RNA. The RNase III activity isolated from C. crescentus cell extracts has potential use in the analysis of specific RNA species because it was found to be more stringent in the recognition of cleavage sites than the E. coli enzyme.
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PMID:Purification and characterization of an RNA processing enzyme from Caulobacter crescentus. 634 87

Bacteriophage lambda transcripts were synthesized in vitro using lambda b2 DNA and Escherichia coli RNA polymerase. RNA molecules initiating with ATP were labeled exclusively at the 5'-end by transcribing in the presence of [gamma-32P]ATP. They were analyzed by electrophoresis in 3.5% polyacrylamide, 7.5 M urea gels followed by autoradiography. The previously known 6 S rho-independent transcript and rho-dependent 9 S and 8 S transcripts were observed. A new rho-dependent 5 S transcript was also detected. The 5 S RNA was not the result of cleavage of larger RNAs by ribonuclease III. Analysis of partial ribonuclease T1 digestion products of isolated 5'-end-labeled transcripts showed that the 5 S RNA is synthesized from promoter pR, the promoter for the 9 S and 8 S RNA species. A similar analysis of transcripts labeled with 32P exclusively at the 3'-terminus by the post-transcriptional addition of a [32P]pCp moiety with T4 RNA ligase revealed the positions of guanosine nucleotides proximal to the 3'-hydroxyl end. This information, and inspection of the known DNA nucleotide sequence downstream from pr, allowed us to conclude that the 5 S RNA is a mixture of molecules 112 and 113 nucleotides long terminating in the middle of gene cro. The nucleotide sequence at the 3'-end of the 5 S RNA is similar to that of other rho-dependent transcripts and lacks the oligo(U) found at the terminus of rho-independent transcripts.
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PMID:Characterization of a rho-dependent termination site within the cro gene of bacteriophage lambda. 644 59

The nucleotide sequence through the transcription termination site for Escherichia coli RNA polymerase at the end of the early region of T7 DNA has been determined. RNA chains terminate at adjacent residues in the DNA sequence: about 2/3 of the chains end in C and 1/3 in G. A potential stem and loop structure, containing a stem of eight uninterrupted base pairs and a four-base loop, is centered 17-18 bases ahead of the chain termini. Transcription by E. coli RNA polymerase terminates efficiently at this site in vivo and in vitro, but transcription by T7 RNA polymerase is essentially unaffected. There are no primary cleavage sites for RNase III near the transcription termination site. The site of termination lies within HpaI fragment Q of T7 DNA, whose entire 446-nucleotide long sequence was determined. Cleavage sites for other restriction endonucleases are located conveniently for manipulating the DNA sequence around the termination site. The coding sequence for the last 82 amino acids of the T7 DNA ligase protein was identified, as was the beginning of a coding sequence for a possible late T7 protein from beyond the termination site.
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PMID:The transcription termination site at the end of the early region of bacteriophage T7 DNA. 700 54

Bacillus subtilis bacteriophage SP82 codes for several early RNAs that were shown previously to be cleaved by an RNase III-like enzyme called "Bs-RNase III." Cloning of DNA fragments that encode these RNA sequences downstream of a T7 RNA polymerase promoter allowed the synthesis of substrates that were used to test the cleavage specificity of Bs-RNase III, which was purified from a protease-deficient strain of B. subtilis. Single nucleotide changes at or near the cleavage site and deletions upstream and downstream of the cleavage site were constructed. The effects of these changes on the rate of Bs-RNase III cleavage were measured. The activity of Bs-RNase III and Escherichia coli RNase III on heterologous substrates was also tested. Although the local environment of the site of Bs-RNase III cleavage appears very similar to that of E. coli RNase III, there are important differences in their substrate specificity.
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PMID:Substrate specificity of an RNase III-like activity from Bacillus subtilis. 752 87

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages. Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNA(AGG) in the cell to become sequestered in translating peptidyl-tRNA(AGG) -mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome of frameshift, hop, or terminate translation.
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PMID:Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system. 767 94

The complex amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of E coli contains important genes for several fundamental cellular processes, including cell-wall hydrolysis (amiB), DNA repair (mutL), tRNA modification (miaA) and proteolysis (hflX-hflK-hflC). We report here the transcriptional pattern and possible posttranscriptional regulation of mutL, miaA and hfq genes of this superoperon. RNase protection analysis of mRNA transcribed from the bacterial chromosome demonstrated that there is co-transcription of mutL and miaA. In addition, two internal promoters, PmiaA and P1hfq were identified and mapped to 201 and 837 nucleotides upstream from the respective translation start sites. PmiaA contains poor matches to the -10 and -35 regions of the sigma-70 RNA polymerase consensus sequences, but it contains multiple potential Fis-binding sites and an upstream AT-rich region with poly(A) sequences. The basic arrangement of Fis-binding sites followed by an AT rich region is shared with promoters for rRNA operons and some of the tRNA and tRNA modification genes. As part of an initial study of mutL and miaA regulation, we measured transcript amounts in isogenic rne, rnc and rne rnc double mutants which are deficient in RNase E, RNase III or both. The amounts of steady state level mutL-miaA cotranscript, PmiaA transcript and P1hfq transcript increased eight-, nine- and three-fold respectively in an rne3071 mutant when compared to the rne+ parent. In contrast, amounts of the three transcripts were the same in an rnc105 mutant and its rnc+ parent. These results indicate that mutL, miaA, and hfq expression could be regulated by multiple mechanisms, including degree of cotranscription from upstream genes, modulation of internal promoter strength, and by RNase E activity. A model is presented for RNase E-mediated posttranscriptional regulation that may coordinate mutL expression with replication and miaA with tRNA amounts under different growth conditions, especially during nutrient upshifts.
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PMID:Transcriptional patterns of the mutL-miaA superoperon of Escherichia coli K-12 suggest a model for posttranscriptional regulation. 774 52


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