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 synthesis, solution conformation, and interaction with DNA of three 8-residue peptides structurally related to the heptad repeat unit found at the C-terminus of RNA polymerase II are reported. Peptides QQ, XQ, and PQ are derived from the parent sequence YSPTSPSY (peptide YY), which was reported to bind to DNA by bisintercalation [M. Suzuki (1990) Nature, Vol. 344, pp. 562-565], and contain either a 2-quinolyl (Q), 2-quinoxolyl (X), or 5-phenanthrolyl (P) group in place of the aromatic side chains of the N- and C-terminal tyrosine residues present in the parent sequence. The combined results of linear dichroism and induced CD measurements of peptides QQ, XQ, and PQ with calf thymus DNA are consistent with weak binding of the peptides to DNA in a preferred orientation in which the chromophores are intercalated. Small increases in the melting temperatures of poly[d(A-T)2] are also consistent with the peptides interacting with DNA. While enzymatic footprinting with DNase I showed no protection from cleavage by the enzyme, chemical footprinting with fotemustine showed that the peptides modify the reactivity of the major groove, presumably via minor groove binding. Peptide QQ inhibited fotemustine alkylation significantly more than either XQ or PQ, and slightly more than YY. In aqueous solution, nmr experiments on QQ, XQ, and PQ show a significant population of a conformation in which Ser2-Pro3-Thr4-Ser5 form both type I and type II beta-turn conformations in equilibrium with open chain conformations. Nuclear magnetic resonance titration experiments of PQ with (GCGTACGC)2 showed small changes in chemical shifts, consistent with the formation of a weak nonspecific complex. Analogous experiments, using peptides QQ and XQ with (GCGTACGC)2, and peptide YY with (CGTACG)2, showed no evidence for the interaction of the peptides with these oligonucleotides. These results show that peptides of general structure XSPTSPSZ are weak nonspecific DNA binders that differ significantly from previously characterized S(T)PXX DNA-binding motifs that are generally AT-selective minor groove binders.
Biopolymers 1997 Oct 05
PMID:DNA-binding studies of XSPTSPSZ, derivatives of the intercalating heptad repeat of RNA polymerase II. 928 89

The question of long-range allosteric transitions of DNA secondary structure and their possible involvement in transcriptional activation is discussed in the light of new results. A variety of recent evidence strongly supports a fluctuating long-range description of DNA secondary structure. Balanced equilibria between two or more different secondary structures, and the occurrence of very large domain sizes, have been documented in several instances. Long-range allosteric effects stemming from changes in sequence or secondary structure over a small region of the DNA have been observed to extend over distances up to hundreds of base pairs in some cases. The discovery that coherent bending strain beyond a threshold level in small (N < or = 250 base pairs (bp)] circular DNAs significantly alters the DNA secondary structure has important implications, especially for transcriptional activators that either bend the DNA directly or are involved in the formation of DNA loops of sufficiently small size (N < or = 250 bp). Whether the RNA polymerase is activated primarily via protein: protein contacts, as is widely believed, or instead via a bend-induced allosteric transition of the DNA in such a small loop, is now an open question. Binding of the transcriptional activator Sp1 to linear DNA induces a remarkably long-range change in its secondary structure, and catabolite activator protein binding to a supercoiled DNA behaves similarly, though possibly for different reasons. Compelling evidence for a bend-induced long-range structural transmission effect of the transcriptional activator integration host factor on RNA polymerase activity was recently reported. These results may augur a new paradigm in which allosteric transitions of duplex DNA, as well as of the proteins, are involved in the regulation of transcription.
Biopolymers 1997
PMID:The question of long-range allosteric transitions in DNA. 959 80

Fluorescence resonance energy transfer (FRET) is a technique allowing measurements of atomic-scale distances in diluted solutions of macromolecules under native conditions. This feature makes FRET a powerful tool to study complicated biological assemblies. In this report we review the applications of FRET to studies of transcription initiation by Escherichia coli RNA polymerase. The versatility of FRET for studies of a large macromolecular assembly such as RNA polymerase is illustrated by examples of using FRET to address several different aspects of transcription initiation by polymerase. FRET has been used to determine the architecture of polymerase, its complex with single-stranded DNA, and the conformation of promoter fragment bound to polymerase. FRET has been also used as a binding assay to determine the thermodynamics of promoter DNA fragment binding to the polymerase. Functional conformational changes in the specificity subunit of polymerase responsible for the modulation of the promoter binding activity of the enzyme and the mechanistic aspects of the transition from the initiation to the elongation complex were also investigated.
Biopolymers
PMID:Fluorescence resonance energy transfer analysis of escherichia coli RNA polymerase and polymerase-DNA complexes. 1198 81

End-initiated transcription of a 256 base-pair (bp) template containing a single uniquely positioned nucleosome by yeast and calf thymus nuclear RNA polymerases II (pol II) was analyzed in vitro. The nucleosome-specific pausing pattern is similar to the pattern observed in the case of transcription of the same nucleosome by yeast RNA polymerase III. However, the pausing pattern is clearly different from the patterns observed previously during transcription by promoter-initiated and assembled pol II. This suggests that end-initiated and promoter-initiated RNA polymerases differ in the way they progress through the nucleosome. The rates of transcription through the nucleosome by pol II are significantly lower than the rates observed in the case of SP6 polymerase and RNA polymerase III. Using calf thymus pol II, we have investigated the possibility that phosphorylation of the C-terminal domain (CTD) facilitates transcription through the nucleosome. The rates of transcription through the nucleosome by phosphorylated (IIO) and nonphosphorylated (IIA) forms of calf thymus pol II are very similar. This suggests that CTD phosphorylation is not sufficient to facilitate transcription through the nucleosome by end-initiated pol II.
Biopolymers 2003 Apr
PMID:Role of C-terminal domain phosphorylation in RNA polymerase II transcription through the nucleosome. 1266 77

T7 RNA polymerase is an enzyme that carries out transcription using DNA as the template and ribonucleotides as the substrates. Here we report the association of the polymerase with 1-anilinonaphthalene-8-sulfonate (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS), which are two fluorescent hydrophobic probes that are frequently used to study structural perturbations in proteins and intermediate states of proteins during folding and unfolding. Our results from the fluorescence titration data show that these two molecules bind to the enzyme with dissociation constants on the micromolar order. The results from the tryptic digestion of the enzyme in the absence and presence of the probes show that they inhibit the rate of tryptic digestion. Circular dichroism spectroscopic studies of the protein in the near UV region indicate that both probes induce tertiary structural changes in the polymerase. There is also a probe (ANS or bis-ANS) induced inhibition of the enzymatic activity. All these results are attributed to association of the probes with the enzyme, leading to an alteration in the conformation of T7 RNA polymerase. This limits the use of these extrinisic probes to the study of the folding properties of the enzyme.
Biopolymers 2003
PMID:Association of fluorescent probes 1-anilinonaphthalene-8-sulfonate and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid with T7 RNA polymerase. 1283 79

Late expression factor 4 (LEF4) is one of the four subunits of Autographa californica nuclear polyhedrosis virus (AcNPV) RNA polymerase. LEF4 was overexpressed in Escherichia coli and recombinant protein was subjected to structural characterization. Chemical induced unfolding of LEF4 was investigated using intrinsic fluorescence, hydrophobic dye binding, fluorescence quenching, and circular dichroism (CD) techniques. The unfolding of LEF4 was found to be a non-two state, biphasic transition. Intermediate states of LEF4 at 2M GnHCl and 4M urea shared some common structural features and hence may lie on the same pathway of protein folding. Steady-state fluorescence and far-UV CD showed that while there was considerable shift in the wavelength of emission maximum (lambda(max)), the secondary structure of LEF4 intermediates at 2M GnHCl and 4M urea remained intact. Further, temperature induced denaturation of LEF4 was monitored using far-UV CD. This study points to the structural stability of LEF4 under the influence of denaturants like urea and temperature. Although LEF4 is an interesting model protein to study protein folding intermediates, in terms of functional significance the robust nature of this protein might reflect one of the several strategies adapted by the virus to survive under very adverse environmental and physiological conditions.
Biopolymers 2009 Jul
PMID:Biophysical characterization and unfolding of LEF4 factor of RNA polymerase from AcNPV. 1927 20

6-Fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is a novel antiviral compound with broad activity against influenza virus and diverse RNA viruses. Its active metabolite, T-705-ribose-5'-triphosphate (T-705-RTP), is recognized by influenza virus RNA polymerase as a substrate competing with GTP, giving inhibition of viral RNA synthesis and lethal virus mutagenesis. Which enzymes perform the activation of T-705 is unknown. We here demonstrate that human hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts T-705 into its ribose-5'-monophosphate (RMP) prior to formation of T-705-RTP. The anti-influenza virus activity of T-705 and T-1105 (3-hydroxy-2-pyrazinecarboxamide; the analog lacking the 6-fluoro atom) was lost in HGPRT-deficient Madin-Darby canine kidney cells. This HGPRT dependency was confirmed in human embryonic kidney 293T cells undergoing HGPRT-specific gene knockdown followed by influenza virus ribonucleoprotein reconstitution. Knockdown for adenine phosphoribosyltransferase (APRT) or nicotinamide phosphoribosyltransferase did not change the antiviral activity of T-705 and T-1105. Enzymatic assays showed that T-705 and T-1105 are poor substrates for human HGPRT having Km(app) values of 6.4 and 4.1 mM, respectively. Formation of the RMP metabolites by APRT was negligible, and so was the formation of the ribosylated metabolites by human purine nucleoside phosphorylase. Phosphoribosylation and antiviral activity of the 2-pyrazinecarboxamide derivatives was shown to require the presence of the 3-hydroxyl but not the 6-fluoro substituent. The crystal structure of T-705-RMP in complex with human HGPRT showed how this compound binds in the active site. Since conversion of T-705 by HGPRT appears to be inefficient, T-705-RMP prodrugs may be designed to increase the antiviral potency of this new antiviral agent.
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PMID:Role of human hypoxanthine guanine phosphoribosyltransferase in activation of the antiviral agent T-705 (favipiravir). 2390 13

Favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) is an anti-viral agent that selectively and potently inhibits the RNA-dependent RNA polymerase (RdRp) of RNA viruses. Favipiravir was discovered through screening chemical library for anti-viral activity against the influenza virus by Toyama Chemical Co., Ltd. Favipiravir undergoes an intracellular phosphoribosylation to be an active form, favipiravir-RTP (favipiravir ribofuranosyl-5'-triphosphate), which is recognized as a substrate by RdRp, and inhibits the RNA polymerase activity. Since the catalytic domain of RdRp is conserved among various types of RNA viruses, this mechanism of action underpins a broader spectrum of anti-viral activities of favipiravir. Favipiravir is effective against a wide range of types and subtypes of influenza viruses, including strains resistant to existing anti-influenza drugs. Of note is that favipiravir shows anti-viral activities against other RNA viruses such as arenaviruses, bunyaviruses and filoviruses, all of which are known to cause fatal hemorrhagic fever. These unique anti-viral profiles will make favipiravir a potentially promising drug for specifically untreatable RNA viral infections.
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PMID:Favipiravir (T-705), a broad spectrum inhibitor of viral RNA polymerase. 2876 16