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)

We have developed general methods for joining together, via cleavable disulfide bonds, either two unprotected polynucleotides or a polynucleotide and a peptide or protein. To join two oligonucleotides, each is first converted to an adduct in which cystamine is joined to the 5'-terminal phosphate of the oligonucleotide by a phosphoramidate bond. The adducts are mixed and reduced with dithiothreitol. The dithiothreitol is then removed by dialysis. Oxidation by atmospheric oxygen occurs to yield the required dimer. To join an oligonucleotide to a cysteine-containing peptide or protein, the 5'-cystamine oligomer is first converted to a 2'-pyridyldisulfide adduct and then reacted with an excess of the peptide or protein. If the peptide does not contain a free cysteine residue, it is first treated with iminothiolane to introduce one or more sulfhydryl groups. We have used these procedures to join a 16 mer deoxynucleotide probe and MDV-1 RNA, a substrate of Q beta RNA polymerase. This adduct hybridizes with a complementary target DNA. We have also joined a 16mer probe to peroxidase and MDV-1 RNA to human IgG. The probe-peroxidase adduct maintains enzymatic activity and the MDV-1 RNA-IgG adduct binds to a complementary anti-IgG.
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PMID:Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds. 337 70

Omega is a small protein found associated with Escherichia coli RNA polymerase. The role of omega, if any, in transcription is not known. We have cloned the omega-encoding gene (rpoZ) so that we can produce large amounts of omega by over-production and to introduce mutations in its gene. We determined the N-terminal amino acid (aa) sequence of omega by aa microsequencing. Using the sequence we synthesized an eight-fold ambiguous 14-mer oligodeoxynucleotide probe and screened an E. coli genomic library using the base composition independent method of hybridization reported by Wood et al. [Proc. Natl. Acad. Sci. USA 82 (1985) 1585-1588]. With this method we isolated a clone that contained part of rpoZ which we used as a probe to isolate the complete gene. The sequence of the region containing the rpoZ gene predicts a highly charged protein of 91 aa with an Mr of 10 105. In addition, upstream from the gene is a good promoter-like sequence. We have verified by S1 mapping that in vivo transcripts originate from this promoter and possibly from a second promoter farther upstream.
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PMID:The cloning and sequence of the gene encoding the omega subunit of Escherichia coli RNA polymerase. 354 61

A DNA oligomer 15 nucleotides long was used to probe the involvement of RNA secondary structure in the control of transcription termination at the attenuator of the tryptophan (trp) operon of Escherichia coli. This 15-mer is perfectly complementary to a segment of trp RNA that is thought to play a role in regulation of attenuation. When added to an in vitro transcription reaction mixture containing wild-type E. coli or Salmonella typhimurium trp operon templates, the complementary 15-mer caused a 4-fold increase in read-through transcription. By contrast, the 15-mer did not affect attenuation when a mutant E. coli template was used that does not allow formation of a crucial RNA secondary structure. Control experiments established that oligomers that were not complementary to E. coli trp leader RNA did not affect attenuation and that the 15-mer did not reduce termination when the transcript lacked a complementary region. Other experiments established that the 15-mer did not increase read-through transcription by allowing RNA polymerase molecules that might have already stopped at the attenuator to resume transcription. These findings provide direct support for the view that alternate base-paired structures control transcription termination at the trp attenuator.
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PMID:Transcription termination at the tryptophan operon attenuator is decreased in vitro by an oligomer complementary to a segment of the leader transcript. 617 92

The aggregation equilibria of Escherichia coli RNA polymerase core and holoenzyme have been studied by velocity sedimentation as a function of [NaCl] both in the presence and in the absence of MgCl2. Effects of other anions (F- and I-), pH, and temperature have also been examined. Diffusion coefficients obtained by quasi-elastic light scattering (QLS) at high and low salt concentrations were used in conjunction with sedimentation coefficients under these conditions to obtain molecular weights of the protomer and aggregates of the core enzyme. At low salt concentration, core aggregates to a tetramer in the absence of MgCl2 and to an octamer in the presence of MgCl2. Some ambiguity exists in the interpretation of the sedimentation and QLS data for holoenzyme. The sedimentation results are consistent with the formation of dimers at low salt, both in the presence and in the absence of MgCl2. In all cases, equilibrium constants were calculated assuming a simple monomer--j-mer stoichiometry. These equilibrium constants are extremely sensitive functions of the concentration and type of monovalent anion. In Cl-, aggregation of both core and holoenzyme begins abruptly when the salt concentration is reduced below approximately 0.2 M (at a protein concentration of approximately 0.30 mg/mL); for core, substitution of I- for Cl- suppresses aggregation while F- enhances aggregation at a fixed anion concentration. No specific effect of monovalent cations (Na+, NH4+) is observed; Mg2+ has no effect on holoenzyme dimerization and has little effect on the salt range of core aggregation, though the stoichiometries of the core aggregates in the presence and absence of Mg2+ differ. Anion effects on these equilibria were modeled by assuming that a class of anion-binding sites on the protomer is not present in the aggregate, so that anion release accompanies aggregation. Analytical expressions for several models of the effect of anions on the aggregation equilibria were derived by using the method of binding polynomials. The salt dependence of the aggregation equilibria in the absence of Mg2+ appears inconsistent with a model in which the anion-binding sites on the protomer are independent (noncooperative), but it is well described by a model in which anion binding to the protomers occurs in a completely cooperative manner. The molecular basis of this apparent cooperative effect of anions on the aggregation equilibria is proposed to be an allosteric effect of anions on conformational equilibria of the protomers of core polymerase and the holoenzyme. Implications of such a salt-dependent conformational transition for the DNA-binding interactions of the enzyme are considered.
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PMID:Aggregation equilibria of Escherichia coli RNA polymerase: evidence for anion-linked conformational transitions in the protomers of core and holoenzyme. 675 71

A group of mutants isolated from E coli K12(tif-1) display a very pleiotropic phenotype. The main characteristic of these mutants, temporarily designated 'S' strains, is their ability to suppress a large number of mutations. High efficiency of suppression is correlated with increased thermolability of cellular proteins, indicating an impairment in the fidelity of protein synthesis. Efficient suppression is also accompanied by the appearance of new characteristics like simultaneous resistance to several antibiotic drugs (Sm, Spc, and Mer), and plasmid-like DNA circles. Genetic studies show that the suppressor character is located in the rpoBC region. In a large number of spontaneous Rifr mutants, isolated from these strains, suppression or resistance to drugs is lost. The findings suggest that the phenotype of 'S' strains is due to an altered RNA polymerase causing erroneous transcription.
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PMID:A novel form of suppression due to an altered RNA polymerase. 701 49

High field 1H-NMR studies of a synthetic 21-mer RNA fragment, corresponding to residues +114 to +134 within the trp leader mRNA transcript, have been carried out. Seven well resolved imino proton resonances corresponding to six C-G and one A-U hydrogen bonded base pairs, together with their characteristic NOE patterns can be identified in the NMR spectrum. This experimental result provides direct evidence for the postulated stem-loop secondary structure, 3:4, which has been reported to act as a transcription termination signal for RNA polymerase.
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PMID:NMR evidence for the RNA stem-loop structure involved in the transcription attenuation of E. coli trp operon. 750 28

The human mdr1 gene encodes a transmembrane glycoprotein the over-expression of which is associated with development of multidrug resistance in human tumor cells. A negative modulation of human mdr1 has been attempted via a 27-mer unmodified triple helix-forming oligonucleotide, named 1D, targeted to a homopurine sequence in the coding region of the gene. By administering 10 microM of 1D we could find a significant reduction in MDR1 mRNA levels in the human drug-resistant cell line CEM-VLB100. This effect appears to be specific and due to a transient block of RNA polymerase mediated by triple helix formation.
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PMID:Effect of unmodified triple helix-forming oligodeoxyribonucleotide targeted to human multidrug-resistance gene mdr1 in MDR cancer cells. 792 5

The temperate phage phi C31 is the most studied bacteriophage infecting Streptomyces spp., and has been used to develop an extensive and widely used series of cloning vectors. The sequence of 10 kb of phi C31 DNA containing most or all of the essential early genes was determined. Among the ORFs, 14 (perhaps 15) appear to be protein-coding, and these have been designated ORF1 to ORF14 and ORFX. Previously mapped transcripts appear to initiate upstream from ORFs 1, 8, 11 and 12, and within ORF3 and ORF12, in each case close to one example of the unusual ('21-mer') sequences that appear to serve as a recognition site for RNA polymerase early in the phi C31 lytic cycle [Ingham et al., Mol. Microbiol. 9 (1993) 1267-1274]. Further copies of the 21-mer are upstream from ORF2 and ORF13. There are four recognisable examples of a conserved inverted repeat sequence motif (CIR) thought to bind phi C31 repressor [Smith and Owen, Mol. Microbiol. 5 (1991) 2833-2844]. Only one CIR is closely associated with a 21-mer sequence, though three are located between known transcription units. Of all 14 ORFs, only one (ORF11) would encode a protein unmistakably resembling other known proteins; its product appears to be a DNA polymerase. Strikingly, two codons, TTA (Leu) and AGG (Arg), are absent from the 14 ORFs.
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PMID:Sequence of the essential early region of phi C31, a temperate phage of Streptomyces spp. with unusual features in its lytic development. 808 46

The vaccinia virus H4 gene encodes an essential subunit of the DNA-dependent RNA polymerase holoenzyme encapsidated within virus particles (Ahn, B., and Moss, B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3536-3540; Kane, E. M., and Shuman, S. (1992) J. Virol. 66, 5752-5762). The role of this protein in transcription of viral early genes was revealed by the effects of affinity-purified anti-H4 antibody on discrete phases of the early transcription reaction in vitro. Anti-H4 specifically prevented the synthesis of a 21-nucleotide nascent RNA chain but had no impact on elongation of the 21-mer RNA by preassembled ternary complexes. Inhibition of initiation but not elongation was also observed with affinity-purified anti-D6 antibody directed against the 70-kDa subunit of the vaccinia early transcription initiation factor (ETF). Native gel mobility-shift assays showed that anti-H4 prevented the NTP-dependent recruitment of RNA polymerase to the preinitiation complex of ETF bound at the early promoter. Two species of ternary complexes could be resolved by native gel electrophoresis. Addition of anti-H4 to preformed complexes elicited a supershift of both ternary species but not of the preinitiation complex. Supeshift by anti-D6 revealed that the more rapidly migrating species of ternary complex did not contain immunoreactive ETF. Loss of ETF from the ternary complex was time-dependent. Thus, whereas the H4 protein was a stable constituent of the elongation complex, ETF was dissociable. We suggest that H4 functions as a molecular bridge to ETF and thereby allows specific recognition of early promoters by the core RNA polymerase. H4 is unlike bacterial sigma factor in that it remains bound to polymerase after the elongation complex is established.
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PMID:A role for the H4 subunit of vaccinia RNA polymerase in transcription initiation at a viral early promoter. 818 18

Polyamide oligomers, termed peptide nucleic acids (PNAs), bind with high affinity to both DNA and RNA and offer both antisense and antigene approaches for regulating gene expression. When a PNA binds to a complementary sequence in a double-stranded DNA, one strand of the duplex is displaced, and a stable D-loop is formed. Unlike oligodeoxynucleotides for which binding polarity is determined by the deoxyribose sugar, the unrestrained polyamide backbone of the PNA could permit binding to a DNA target in an orientation-independent manner. We now provide evidence that PNAs can, in fact, bind to their complementary sequence in DNA independent of the DNA-strand polarity--that is, a PNA binds to DNA in both "parallel" and "antiparallel" fashion. With a mixed-sequence 15-mer PNA, kinetic studies of PNA.DNA interactions revealed that D-loop formation was rapid and the complex was stable for several hours. However, when measured either by gel-mobility-shift analysis or RNA polymerase II-elongation termination, D-loop formation was salt dependent, but PNA-strand dissociation was not salt dependent. We observed that D-loop-containing DNA fragments had anomalous gel mobilities that varied as a function of the position of the D-loop relative to the DNA termini. On the basis of permutation analysis, the decreased mobility of the PNA.DNA complex was attributed to a bend in the DNA at or near the D-loop.
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PMID:Strand-invasion of duplex DNA by peptide nucleic acid oligomers. 824 56


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