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 nucleocapsid protein (NC) of all animal retroviruses, encoded by the gag gene, is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. NC is also thought to play one or more accessory roles in reverse transcription. Mature NC (p7NC) from human immunodeficiency virus type 1 (HIV-1) is a 71-amino acid, basic protein which contains two Cys3His Zn(II) retroviral-type zinc finger domains. Herein, we describe the subcloning and expression of HIV-1 NC, denoted NC71, from an inducible phage T7 RNA polymerase promoter in Escherichia coli. Purified NC71 can be reversibly reconstituted with 2 g.at Zn(II) determined by atomic absorption. Ultraviolet circulation dichroism spectroscopy has been used to characterize the complexes between highly purified NC71 and the RNA homopolynucleotide poly(A) and E. coli tRNA(mixed). On poly(A), Zn2 NC71 is characterized by an apparent site size n = 15 +/- 3 nucleotides and high affinity (Kapp = 3 x 10(7) M-1) and moderately cooperative (omega approximately 170 +/- 25) binding. A mixture of E. coli tRNA species (tRNA(mixed) was used to probe the conformational changes induced in tRNA upon binding of HIV-1 NC71. Two structural forms of tRNA(mixed), which differ in their degree of tertiary structure, were assayed for their susceptibility to denaturation by NC71. Five molar monomer equivalents of NC71 are required to denature the "inactive" tRNA in the absence of Mg2+. A Zn(II)-free, oxidized form of NC71 was also shown to unwind inactive tRNA with the same efficiency and stoichiometry. The detailed spectral changes which occur on NC-induced denaturation closely mimic temperature-induced denaturation of inactive tRNA(mixed). The prototype helix-destabilizing protein, T4 gene 32 protein, is unable to unwind this form of tRNA under the same conditions. The stoichiometry of unwinding of inactive tRNA by NC71 is consistent with the site size determined with poly(A). An "active" form of tRNA(mixed), prepared by thermal denaturation and refolding of the inactive form with Mg2+, proved less susceptible to both temperature and NC71-induced unwinding. The mechanistic implications of these findings on the reported biochemical activities of RNA:RNA annealing and replication primer tRNA positioning by NC are discussed.
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PMID:Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA. 155 77

We investigated the accuracy of the insertion process in RNA chain elongation catalyzed by wheat germ RNA polymerase II. Error frequencies varied from 1 misinserted nucleotide per 250 polymerized correct substrates to less than 1 in 2 x 10(5), depending on template sequence and nature of the divalent metal cofactor. Higher error ratios were observed in the presence of Mn2+ compared to Mg2+, and with alternating poly[d(G-C)].poly[d(G-C)] compared to poly[d(A-T)].poly[d(A-T)]. In this latter case the eukaryotic RNA polymerase was as accurate as Escherichia coli RNA polymerase holo-enzyme. The fidelity of wheat germ RNA polymerase II was also examined in transcription of polynucleotide templates in the poly[d(G-C)] family adopting either the right-handed B or left-handed Z conformations. Error ratios for noncomplementary ATP increased markedly under experimental conditions favoring the B-to-Z conformational transition of the alternating copolymers. In accordance with the results of previous studies, the rate of productive elongation, i.e. the synthesis of poly[r(G-C)], was depressed, suggesting that the decreased accuracy of the enzyme derived from an altered competence of the enzyme to form elongation complexes on the left-handed DNA. As judged by the large difference in apparent Km values of the enzyme for complementary and noncomplementary nucleoside triphosphates, part of the discrimination between substrates seemed to take place at the initial binding step. Furthermore, the results indicate that wheat germ RNA polymerase II was able to elongate a primer with a 3'-terminal mismatch, and thus to incorporate the mismatched nucleotide stably in the nascent RAN. However, the probability of productive RNA chain elongation was much lower with noncognate than with the complementary substrates.
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PMID:Accuracy of wheat-germ RNA polymerase II. General enzymatic properties and effect of template conformational transition from right-handed B-DNA to left-handed Z-DNA. 158 82

Reverse transcriptase has been purified from feline immunodeficiency virus (FIV) by DEAE-cellulose and phosphocellulose chromatography. The purified enzyme consists of a single protein with a Mr of 67,000. When proteolysis is not minimized during purification, a fragment of Mr 54,000 is also observed. This is similar to the reverse transcriptase from human immunodeficiency virus type 1 (HIV), which consists of a polypeptide of Mr 66,000; when proteolysis is not minimized during purification, a fragment of Mr 51,000 is also observed. In direct comparisons, the FIV reverse transcriptase is very similar to the HIV reverse transcriptase in template specificity and requirements for Mg2+. In contrast to these similarities, the FIV and HIV reverse transcriptases are substantially different in primary sequence, as determined by peptide mapping.
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PMID:Characterization of reverse transcriptase from feline immunodeficiency virus. 169 Jul 35

We have devised a method to follow the stability of individual ternary transcription complexes containing Escherichia coli RNA polymerase halted at many different sites along a DNA template during the transcription process. Studies of complexes formed with phage T7 DNA templates reveal at least three general classes of ternary complexes that differ dramatically in their properties. Complexes of one sort (normal complexes) are highly stable to dissociation and denaturation under a variety of solution conditions. They remain intact and active for up to 24 hours even in salt concentrations up to 1 M-K+. This suggests that they are stabilized to a significant extent by non-ionic interactions between RNA polymerase and the nucleic acids. We consider these to be the normal complexes formed during RNA chain elongation. Complexes of a second sort (release complexes) dissociate rapidly, releasing free RNA transcripts and active RNA polymerase. The rate of dissociation is substantially enhanced by elevated concentrations of K+, hence the interaction between RNA polymerase and nucleic acids in these complexes is stabilized predominantly by ionic interactions. However, release complexes are stabilized by millimolar concentrations of Mg2+, which as been implicated in stabilization of the binding of RNA to free RNA polymerase. These complexes are formed at DNA sequences that we refer to as release sites. Analysis of DNA sequences at release sites reveals that all share a common feature, the potential to form an RNA hairpin in the region just upstream from the actual 3' end of the released RNA. Experiments incorporating IMP in the transcript and blocking potential hairpin formation with DNA oligomers support a direct role for an RNA hairpin in triggering the release reaction. Changes in the 3'-proximal DNA sequences generally have little effect on the presence or rate of the release reaction, although there are significant exceptions. The results suggest that the presence of certain RNA hairpins in the region six to ten nucleotides upstream from the transcript growing point can trigger a substantial structural transition in the ternary transcription complex, forming a "release mode" complex from which transcript dissociation is facilitated. This release, mode complex may be a central intermediate in RNA chain termination. A final class of complexes (dead-end complexes) appear to be elongating complexes that have entered a state or conformation that is stable, but is blocked in resuming the normal elongation reaction. Such complexes bear active RNA polymerase, and can be restarted after limited pyrophosphorolysis. The structural elements that determine the formation of dead-end complexes are not yet known.
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PMID:RNA chain elongation by Escherichia coli RNA polymerase. Factors affecting the stability of elongating ternary complexes. 169 94

Reverse transcriptase (RT) transcribes viral RNA into DNA to be integrated into the host genome. To study epidemiological aspects of human leukemias and lymphomas which are known to express retroviruses, clinical specimens in this report were assayed for divalent cation-dependent viral-specific RT. The assay was carried out with cells solubilized with a detergent to release RT enzyme. RT was purified with poly(U)-Sepharose which fixed all DNA polymerases and assayed with 4 synthetic homopolymers, oligonucleotide primed-templates, poly(rA)-oligo(dT)12-18 or poly(dA)-oligo(dT)12-18 with Mg2+, poly(rC)-oligo(dG)12-18 or poly(rCm)-oligo(dG)12-18 with Mn2+ as divalent cation and [methyl-3H]thymidine 5'-triphosphate or deoxy[8-3H]guanosine 5-triphosphate respectively. Radioactivity incorporation of the precipitate allows quantitation of RT activity. One Hodgkin's disease, one out of 2 B lymphomas, one out of 2 T lymphomas, eight out of 12 leukemias were found to be positive for RT activity as well as acquired immunodeficiency syndrome (AIDS) patients, known to express RT. The obtained RT activity in hematological malignancies was found to be comparable to positive controls such as RT enzymes purified from avian myeloblastosis and Moloney murine leukemia viruses.
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PMID:Presence of reverse transcriptase in human leukemias and lymphomas. 170 70

The DNA-dependent RNA polymerase (EC 2.7.7.6) from Clostridium acetobutylicum DSM 1731 has been purified to homogeneity and characterized. The purified enzyme was composed of four subunits and had a molecular mass of 370,000 Da. Western immunoblot analysis with polyclonal antibodies against the sigma 70 subunit of Escherichia coli RNA polymerase identified the 46,000-Da subunit as an immunologically and probably functionally related protein. The other three subunits of 128,000, 117,000, and 42,000 Da are tentatively analogous to the beta, beta', and alpha subunits, respectively, of other eubacterial RNA polymerases. The RNA polymerase activity was completely dependent on Mg2+, nucleoside triphosphates, and a DNA template. The presence of Mg2+ or Mn2+ in buffers used for purification or storage caused irreversible inactivation of the RNA polymerase.
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PMID:Purification and characterization of the DNA-dependent RNA polymerase from Clostridium acetobutylicum. 170 30

Ternary complexes of RNA polymerase, bearing the nascent RNA transcript, are intermediates in the synthesis of all RNAs and are regulatory targets of factors that control RNA chain elongation and termination. To study the catalytic and regulatory properties of RNA polymerases during elongation, we have developed methods for the preparation of these intermediates halted at defined positions along a DNA template. To our surprise, some of these halted complexes undergo a reaction in which the RNA transcript is cleaved up to 10 nucleotides from its 3'-terminal growing point. The 5'-terminal fragment, bearing a free 3'-OH residue, remains bound to the RNA polymerase-DNA complex and can resume elongation, whereas the 3'-terminal oligonucleotide of 2-10 nucleotides, bearing a 5'-phosphate, is released. RNA cleavage occurs only in the ternary complex and requires a divalent metal ion such as Mg2+. Since RNA polymerases are believed to have a single catalytic site for nucleotide addition, this reaction is unlikely to be due to hydrolysis catalyzed by this site comparable to the 3'----5' exonuclease activity associated with the catalytic center found for some DNA polymerases. Nor is this reaction easily explained by models for transcription elongation that postulate a 12-base-pair DNA.RNA hybrid as intermediate. Instead, we suggest that this is an unusual kind of protein-facilitated reaction in which tight binding of the RNA product to the enzyme strains the RNA phosphodiester linkage, resulting in cleavage of the RNA well away from the catalytic center. By this model, the nascent RNA enters a product binding site beginning 3 or 4 nucleotides from the growing point at the 3' terminus. This RNA binding site extends for up to 16 nucleotides along the protein surface. The stress brought about by this binding appears to vary considerably for different ternary complexes and may play a role in driving the translocation of the RNA polymerase along the DNA.
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PMID:Spontaneous cleavage of RNA in ternary complexes of Escherichia coli RNA polymerase and its significance for the mechanism of transcription. 171 68

The miscoding potential of N2,3-ethenoguanine (epsilon G), one of the carcinogen vinyl chloride adducts to DNA bases, has been evaluated in an Escherichia coli DNA-dependent RNA polymerase in vitro system. Epsilon G present in poly(C) templates causes incorporation of cytosine (C), uridine (U) and adenosine (A) under competitive and non-competitive conditions, and in the presence of either Mn2+ and Mg2+ cations, indicating that this modified base still retains the coding properties of unmodified G and can also act as A or U. The formation of hydrogen bonded pairs between different tautomeric forms of epsilon G and C, U and A is proposed. The possible role of epsilon G, along with a role of other vinyl chloride adducts in causing of GC----AT transitions, the most frequent mutation induced by a vinyl chloride metabolite, is discussed.
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PMID:Miscoding potential of N2,3-ethenoguanine studied in an Escherichia coli DNA-dependent RNA polymerase in vitro system and possible role of this adduct in vinyl chloride-induced mutagenesis. 179 43

To investigate the relationship between transcription and polyadenylation, an in vitro system has been developed in which endogenously transcribed pre-mRNAs containing functional polyadenylation sites are rapidly and accurately cleaved in a HeLa nuclear extract. Cleavage of endogenously transcribed substrates differed from that of exogenous substrates in that a proximal 3' terminus was not required, the reaction was more tolerant of increased Mg2+ levels, and endogenous substrates were cleaved more efficiently. A promoter dependence for this reaction was suggested by the observation that substrates transcribed by bacteriophage T7 RNA polymerase in the presence of nuclear extract were not cleaved. In addition, analysis of the bovine growth hormone poly(A) site indicated that it is highly efficient in vitro which agrees with previous in vivo data. The availability of an in vitro system in which transcription and polyadenylation are coupled should facilitate analysis of the relation between 3' end processing and RNA polymerase II transcription termination as well as the promoter requirements for polyadenylation.
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PMID:Coupled transcription-polyadenylation in a cell-free system. 191 66

We have developed a T7 RNA polymerase based transcription system for the production of fully complementary RNA molecules (i.e., molecules capable of forming blunt-ended duplex species) as the direct products of transcription, thus rendering unnecessary the enzymatic removal of single-stranded ends. A combined gel electrophoretic and hydrodynamic analysis of a 180 bp double-stranded (ds) RNA molecule containing four A5-tracts in approximate phase coherence with the helix repeat provides no indication that the helix axis is curved, in sharp contrast to DNA molecules containing phased A-tracts. The electrophoretic behavior of dsRNA molecules reveals that their mobilities in nondenaturing acrylamide gels are approximately 10-20% lower than the corresponding mobilities of duplex DNA, in accord with earlier observations in the literature. Furthermore, the relative mobilities are only slightly modulated by gel concentration, the concentration of monovalent salt, or the presence of spermidine and/or Mg2+. The reduced mobilities are not caused by increased contour length, since direct hydrodynamic measurements using transient electric birefringence indicate that the average helix rise, h, of the dsRNA molecules examined in the current study is 2.8 +/- 0.1 A/bp. The reduced electrophoretic mobilities, extrapolated to zero acrylamide concentration, are consistent with the lower residual charge predicted for dsRNA by counterion condensation theory. Finally, birefringence measurements indicate that dsRNA is only marginally stiffer than DNA, with a persistence length of ca. 500-700 A.
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PMID:Electrophoretic and hydrodynamic properties of duplex ribonucleic acid molecules transcribed in vitro: evidence that A-tracts do not generate curvature in RNA. 202 19


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