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)

Several in vitro properties of partially purified form II RNA polymerase from Drosophila melanogaster embryo nuclei are described. The enzyme preparation is free from contaminating RNase, protein kinase, and polyphosphate kinase activities and can be used to study the incorporation of gamma-32P-labeled nucleoside triphosphates. The enzyme exhibits a biphasic heat inactivation pattern which is probably related to differential lability of its two subforms. However, a considerable protection against heat inactivation is provided by the nucleoside triphosphates present in the in vitro reaction system such that the enzyme catalyzes RNA synthesis in a nearly linear mode for over 2 hr at 30 C. Two initiation inhibitors, rifamycin AF/013 was found unsuitable for critical studies because of the high concentrations necessary for total inhibition (200 micrograms/ml) and particularly because of the obligate use of solvents which secondarily have a destabilizing effect on native DNA. Poly[I] was found to effectively block initiation at very low concentrations (1 microgram/ml). The enzyme rapidly forms poly[I]-resistant preinitiation complexes on both double- and single-stranded DNA. These complexes decay with a half-life of 2.5--3 min. RNA synthesis from poly[I]-resistant complexes amounts to 10% of the total potential synthesis on both double- and single-stranded DNA. Enzyme-DNA saturation experiments indicate that the form II enzyme discriminates two types of sites on Drosophila DNA, tight binding and weak binding, from which RNA synthesis proceeds slowly and rapidly, respectively. The tight-binding sites appear to be analogous to those sites with which the enzyme is able to form poly[I]-resistant complexes.
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PMID:Form II DNA-dependent RNA polymerase from Drosophila melanogaster: general in vitro catalytic properties and template interactions. 11 Mar 17

The in vitro incorporation of gamma-32P-labeled nucleoside triphosphates into RNA by Drosophila melanogaster form II RNA polymerase from template sites which afford protection from the initiation inhibitor, polyriboinosinic acid (poly [I]), is used as a method for enumerating a specific class of transcription initiation sites on D. melanogaster DNA. Such sites number about 4000 per haploid genome for D. melanogaster. This value is in good agreement with the number of functional genetic units in the D. melanogaster genome as determined by classical cytogenetics.
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PMID:Enumeration of drosophila form II DNA-dependent RNA polymerase initiation sites on Drosophila DNA. 11 Mar 18

To determine the special feature of ribosomal RNA promoters that might account for the highly efficient and regulated synthesis of rRNA in E. coli, we have analyzed the beginnings of two ribosomal RNA operons, rrnD and rrnX. DNA sequences for 425 bp preceding those specifying mature 16s rRNA are reported. In vitro transcription of restriction endonuclease fragments containing this region from either operon reveals the presence of two promoters about 110 nucleotides apart; they are denoted P1 and P2. RNA synthesis from P1 is initiated with GTP at position -284 (relative to 16s sequences) in rrnD and with ATP at position -285 in rrnX. At P2, the RNA starts with CTP primarily at position-176 in both operons. The DNA sequences of the two operons are identical for 231 bp preceding the 16s rDNA (including a substantial region around P2); they then diverge almost completely, except for a notable 18 bp homology just preceding the transcription start site for P1. Certain sequences implicated in the recognition of promoters by E. coli RNA polymerase are clearly identifiable in both P1 and P2; other features include an extended region preceding P1 which is strikingly rich in AT base pairs. Possible mechansims by which these tandem promoters contribute to the high frequency of rRNA transcription and to the differential expression of the E. coli rrn operons are discussed.
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PMID:Tandem promoters direct E. coli ribosomal RNA synthesis. 11 Apr 60

We have devised a new procedure for the purification of highly active preparations of Bacillus subtilis RNA polymerase holoenzyme. A column of heparin-agarose A-15m is used to rapidly and quantitatively adsorb RNA polymerase from the initial crude extract fraction. This affinity procedure obviates the necessity of including nucleic acid precipitation or partitioning steps and allows for rapid separation of RNA polymerase from proteolytic activity. The enzyme is further purified by preparative glycerol gradient centrifugation resulting in an overall purification in 200-fold in 24 h with near quantitative recovery of polymerase protein and activity. RNA polymerase holoenzyme is obtained by chromatography on single-stranded DNA-agarose. The in vitro transcription products made by purified preparations of B. subtilis and Escherichia coli RNA polymerase holoenzymes in response to B. subtilis phage phi 29 DNA have been analyzed, and an in vitro transcription map is presented. The E. coli RNA polymerase holoenzyme initiates transcription from three promoter sites not efficiently utilized by the B. subtilis holoenzyme under optimal conditions for RNA synthesis.
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PMID:Purification of Bacillus subtilis RNA polymerase with heparin-agarose. In vitro transcription of phi 29 DNA. 11 9

Escherichia coli and Bacillus subtilis RNA polymerase have almost identical transcription specificities on bacteriophage SPO1 DNA when assayed in a coupled transcription-translation cell free system. SPO1-modified B. subtilis RNA polymerase has altered transcription specificity. It is shown that rifampicin-inhibited E. coli RNA polymerase can completely block transcription of SPO1 DNA by rifampicin resistant B. subtilis enzyme, whereas it has no effect on transcription by SPO1-modified B. subtilis RNA polymerase. We conclude that the new transcription by SPO1-modified RNA polymerase results from newly recognized promoters, rather than by elongation of transcripts which could also be made by B. subtilis vegetative RNA polymerase.
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PMID:The nature of transcription selectivity of bacteriophage SPO1-modified RNA polymerase. 11 44

The relationship between sigma (sigma) and delta (delta) factors of Bacillus subtilis RNA polymerase has been analyzed during initiation of RNA synthesis. When core enzyme (E) containing delta factor (E delta) binds to DNA, the delta factor is released with the formation of an E-DNA complex. The addition of sigma to the E-DNA complex results in the formation of a stable E sigma-DNA complex which can synthesize RNA upon addition of nucleoside triphosphates. Sigma factor, significantly, is not released from the core during RNA synthesis. These results suggest that delta and sigma factors can act sequentially during initiation of RNA synthesis with delta acting as a DNA recognition factor and sigma acting as an initiation factor. The results do not preclude the possibility that E sigma can initiate RNA synthesis correctly since E sigma alone can bind to DNA and initiate RNA synthesis.
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PMID:Sigma factor is not released during transcription in Bacillus subtilis. 11 45

The DNA polymerase of early embryos of Drosophila melanogaster has been purified to near-homogeneity. The purified enzyme gave a single, catalytically active protein band after polyacrylamide gel electrophoresis, under nondenaturing conditions. Four polypeptides with molecular weights 43,000, 46,000, 58,000, and 148,000 were resolved when this band was electrophoresed under denaturing conditions. At high ionic strengths, the DNA polymerase had a sedimentation coefficient of 8.7 S, a Stokes radius of 78 A and frictional ratio of 1.81, parameters that yield a molecular weight of 280,000. The purified DNA polymerase possessed no detectable endo- or exodeoxyribonuclease, ATPase, or RNA polymerase activity. Using an "activated" DNA template-primer, the enzyme had a pH optimum of 8.5. It was stimulated by (NH4)2SO4, KCl, and to a lesser extent, NaCl. A divalent metal cation was absolutely required; MgCl2 stimulating activity 7-fold more than MnCl2. It was inhibited by low concentrations of N-ethylmaleimide and Aphidicolon. Thus the DNA polymerase of D. melanogaster resembles most closely the alpha-DNA polymerases that have been purified from mammalian cells.
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PMID:A high molecular weight DNA polymerase from Drosophila melanogaster embryos. Purification, structure, and partial characterization. 11 15

By using a modification of the BAC spreading method for mounting the DNA for electron microscopy, partial denaturation maps of protein-free phi 29 DNA and of phi 29 DNA containing protein p3 were obtained. In phi 29 p3-DNA1 the protein does not seem to influence the melting of the ends of the molecules. The comparison of the partial denaturation map and the B. subtilis RNA polymerase binding sites indicates that five of the seven early promoters (A1, A2, A3, B2 and C2) are located in A-T rich DNA regions whereas the other two early promoters (B1 and C1) are located in less A-T rich sites.
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PMID:Comparison of the A-T rich regions and the Bacillus subtilis RNA polymerase binding sites in phage phi 29 DNA. 11 82

Fidelity of preribosomal RNA transcription in vitro was studied after selective deproteinization of nucleoli using either sequential salt extraction or sodium deoxycholate treatment. Homochromatography fingerprinting and identification of marker oligonucleotides from a T1 ribonuclease digest of the transcripts were used to evaluate the RNA products. These studies indicated that: (1) nucleoli retained their endogenous RNA polymerase I activity and the specificity of transcription up to 0.6 M NaCl extraction; (2) exogenous RNA polymerase I transcribed nucleolar chromatin only after 1.0 M NaCl extraction and the transcription pattern, like that of totally deproteinized DNA, was completely random; (3) extraction of nucleoli with deoxycholate resulted in a DNP complex in which the endogenous RNA polymerase I transcribed pre-rRNA specifically; however, it also initiated random transcription, producing a "mixed" fingerprint pattern on the homochromatogram. The random transcription was selectively inhibited either by deoxycholate or rifampicin AF/013. These studies indicate that the selectivity of pre-rRNA transcription is due both to the endogenous RNA polymerase I molecules that were involved in transcription in vivo and are tightly bound to the template and to factors in intact nucleoli which prevent random transcription by the released RNA polymerase I molecules.
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PMID:Studies on the specificity of preribosomal RNA transcription in nucleoli after selective deproteinization. 11 95

Gene fusions constructed in vitro have been used to examine transcription regulatory signals from the operon which encodes ribosomal proteins L10 and L7/12 and the RNA polymerase beta and beta' subunits (the rplJL-rpoBC operon). Portions of this operon, which were obtained by in vitro deletions, have been placed between the ara promoter and the lacZ gene in the gene-fusion plasmid pMC81 developed by M. Casadaban and S. Cohen. The effect of the inserted DNA segment on the expression of the lacZ gene (in the presence and absence of arabinose) permits the localization of regulatory signals to discrete regions of the rplJL-rpoBC operon. An element that reduces the level of distal gene expression to one-sixth is located on a fragment which spans the rplL-rpoB intercistronic region. This strongly supports the idea that there is an attenuator in this region. The terminator for the operon is located on a fragment which spans the 3' end of the rpoC gene. The major promoter for the operon precedes the rplJ gene [Yamamoto, M. & Nomura, M. (1978) Proc. Natl. Acad. Sci. USA 75, 3891-3895 and Linn, T. & Scaife, J. (1978) Nature (London) 276, 33-37] and was not examined in this study. However, a weak promoter is observed on the fragment that spans the rplJ-rplL intercistronic region. Other regions of the operon may also contain weak promoters. The contribution of these elements to the regulation of this complex operon is discussed.
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PMID:Control features within the rplJL-rpoBC transcription unit of Escherichia coli. 11 24


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