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 promoters of several eukaryotic genes transcribed by RNA polymerase II contain elements located downstream of the transcriptional start site. To gain insight into how these elements function in the formation of an active transcription complex, we have cloned and sequenced the cDNA that encodes delta, a protein that binds to critical downstream promoter elements in the mouse ribosomal protein rpL30 and rpL32 genes. Our results revealed that the delta protein contains four C-terminal zinc fingers, which are essential for its DNA binding capability and a very unusual N-terminal domain that includes stretches of 11 consecutive negatively charged amino acids and 12 consecutive histidines. The sequence of the delta protein was found to be essentially identical to a concurrently cloned human transcription factor that acts both positively and negatively in the context of immunoglobulin enhancers and a viral promoter. Our structural modeling of this protein indicates properties that could endow it with exquisite functional versatility.
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PMID:Delta, a transcription factor that binds to downstream elements in several polymerase II promoters, is a functionally versatile zinc finger protein. 194 4

The DNA-dependent RNA polymerase containing two intrinsic cobalt ions (Co2-RPase) instead of the naturally occurring zinc was purified from Escherichia coli cells grown in zinc-depleted, cobalt-enriched media. Longitudinal NMR relaxation rates of the H2 and H8 protons of ATP were measured in the absence and presence of up to 92 microM Co2-RPase. No enhancement of the proton relaxation rates was observed in the presence of cobalt-containing enzyme, suggesting that the ATP substrate does not undergo rapid exchange at a site close to either of the intrinsic cobalt ions. This result is in contrast to that previously observed when Co2+ was incorporated into RPase by an in vitro procedure involving partial urea denaturation of the protein.
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PMID:1H NMR study of the interaction of ATP with Escherichia coli RNA polymerase containing in vivo-incorporated Co(II). 195 44

The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae. Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190. We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence. We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I. First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S. cerevisiae. Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S. cerevisiae. Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter. When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis. We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases. From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit.
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PMID:Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I. 199 Feb 81

The successful use of mixed heterologous in vitro transcription systems has suggested that the species specificity of RNA polymerase III transcription is low. To see if this extends to lower eukaryotic class III transcription factors, we compared the interactions of the two yeast assembly factors, TFIIIA and TFIIIC/tau factor, with a homologous yeast 5S rRNA gene and a heterologous Xenopus laevis somatic 5S rRNA gene. Transcription assays showed that the Xenopus gene was transcriptionally inactive in a crude cell-free yeast extract that actively transcribes the homologous gene. However, the Xenopus gene was still able to compete for limiting transcription factors. Electrophoretic DNA binding assays revealed that while TFIIIA bound avidly to the yeast gene (generating the 'A-complex'), it had no affinity for the Xenopus 5S rRNA gene. Nevertheless, a complex of both TFIIIA and TFIIIC/tau factor (the 'AC-complex') was formed on the two genes with similar affinity, although only the complex assembled on the homologous gene was able to activate transcription. Thus enough sequence information is present on the heterologous gene to direct transcription factor assembly, but not to activate transcription. Like its counterpart in Xenopus, the yeast TFIIIA appears to be a zinc binding protein that is inactivated by EDTA and 1,10-phenanthroline, and reactivated in the presence of zinc ions. Bound to the 5S rRNA gene, TFIIIA is however significantly more resistant to inactivation by chelators than in its free state. The AC-complex differs from the A-complex by being less affected by chelators, and by being more sensitive to the dissociating effect of single-stranded DNA.
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PMID:Yeast TFIIIA + TFIIIC/tau-factor, but not yeast TFIIIA alone, interacts with the Xenopus 5S rRNA gene. 201 29

The helper dependence of satellite phage P4 superimposes an additional set of regulatory interactions on those required for the independent maintenance of P4 or its helpers. These interactions allow P4 to exploit a helper phage under a variety of circumstances and can affect expression of the immunity functions and late genes of both phages. The phage P2 lysis/lysogeny decision involves two competing repressors regulating mutually exclusive promoters in the early control region. In the absence of a helper phage, the P4 immunity function plays a role in the choice between lysogeny or the multicopy plasmid state. No evidence exists for a P4-encoded immunity repressor; in P4-lysogenic cells, expression of the P4 DNA replication gene alpha appears to be prevented by premature termination of transcription. Immunity-independent expression of alpha in the multicopy plasmid state involves initiation of transcription at an alternative upstream promoter that is positively regulated by P4 delta protein; the same promoter is activated by P2 Cox protein during derepression of P4 by P2. The mechanism of derepression of P2 by P4 remains to be determined, and the relationship between the P4 immunity and derepression functions and the mutations that allow P4 to grow with a P3 prophage helper is an intriguing area for further exploration. Expression of P2 and P4 late genes is regulated by phage-encoded, zinc-binding transcriptional activators that appear to interact directly with the alpha subunit of RNA polymerase of E. coli. Stimulation of P2 late transcription by P2 Ogr protein depends upon phage DNA replication, whereas activation of transcription from the same promoters by the related P4 delta gene product is replication-independent. Elucidation of the mechanisms underlying these interactions promises to provide new insights into strategies for control of gene expression.
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PMID:Interactions between satellite bacteriophage P4 and its helpers. 208 76

The two substrates between which an internucleotide bond is formed in RNA synthesis occupy two subsites, i and i + 1, on the active site of Escherichia coli RNA polymerase, and each subsite is associated with a metal ion. These ions are therefore useful as probes of substrate interaction during RNA synthesis. We have studied interactions between the metals by EPR spectroscopy. The Zn(II) in the i site and the Mg(II) in the i + 1 site were substituted separately or jointly by Mn(II). The proximity of the metals was established by EPR monitoring of the titration at 5.5 K of the enzyme containing Mn(II) in i with Mn(II) going into the i + 1 site, and the 1:1 ratio of the metals in the two sites was confirmed in this way. The distance between the two metals was determined by EPR titration at room temperature of both the enzyme containing Zn(II) in i and Mn(II) in i with Mn(II) going into the i + 1 site, making use of the fact that EPR spectra are affected by dipolar interactions between the metals. The distances calculated in the presence of enzyme alone, in the presence of enzyme and two ATP substrates, and when poly(dAdT).poly(dAdT) was added to the latter system ranged from 5.2 to 6.7 A.
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PMID:Structural studies on the active site of Escherichia coli RNA polymerase. 1. Interaction of metals on the i and i + 1 sites. 216 69

Since a major function of RNA polymerase must be to bring together substrates in the optimal configuration for internucleotide bond formation, studies have been undertaken to understand the geometrical relationship of the two substrates. A model has been constructed for the geometry of interaction of two ATP molecules poised on the active site of the Escherichia coli enzyme for the formation of the first bond in RNA synthesis. The model is based primarily on the distance, measured by EPR, between the two metals in the i and i + 1 subsites, as well as distances, measured by NMR, from each metal to points on the substrate in the same subsite, in the presence of a poly(dAdT).poly(dAdT) template. Both the Zn(II) in the i site and the Mg(II) in i + 1 are displaced by Mn(II). The nucleotide bases are not parallel to each other, in line with the reaction of the ATP molecules with DNA within the transcription bubble. The metal in the i site appears too far removed from substrate to participate in catalysis, but the metal in i + 1 is in position to bind to the beta- and gamma-phosphate groups and probably is involved in cleavage of the triphosphate, as has been previously suggested.
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PMID:Structural studies on the active site of Escherichia coli RNA polymerase. 2. Geometrical relationship of the interacting substrates. 216 70

The MerR metalloregulatory protein is a heavy-metal receptor that functions as the repressor and Hg(II)-responsive transcription activator of the prokaryotic mercury-resistance (mer) genes. We demonstrate that this allosterically modulated regulatory protein is sensitive to HgCl2 concentrations of 1.0 +/- 0.3 x 10(-8) M in the presence of 1.0 x 10(-3) M dithiothreitol for half-maximal induction of transcription of the mer promoter by Escherichia coli RNA polymerase in vitro. Transcription mediated by MerR increases from 10% to 90% of maximum in response to a 7-fold change in concentration of HgCl2, consistent with a threshold phenomenon known as ultrasensitivity. In addition, MerR exhibits a high degree of selectivity. Cd(II), Zn(II), Ag(I), Au(I), and Au(III) have been found to partially stimulate transcription in the presence of MerR, but concentrations at least two to three orders of magnitude greater than for Hg(II) are required. The molecular basis of the ultrasensitivity and selectivity phenomena are postulated to arise from the unusual topology of the transcription complex and a rare trigonal mercuric ion coordination environment, respectively. This mercuric ion-induced switch is to our knowledge the only known example of ultrasensitivity in a signal-responsive transcription mechanism.
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PMID:Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex. 218 94

Gene 32 protein (g32P), the replication accessory protein from bacteriophage T4, is a zinc metalloprotein which binds with high cooperativity to single-stranded (ss) nucleic acids. The basic N-terminal 21 amino acids (termed the "B" domain) is required for highly cooperative (omega approximately 500) binding of g32P monomers to ss nucleic acids. As part of our studies to systematically evaluate the structural features of the B domain important for cooperative binding, a homogeneous source of g32P which binds noncooperatively to nucleic acids (omega = 1) and is devoid of contamination by native g32P is needed. Herein, we describe large-scale overexpression and purification of recombinant g32P lacking the tryptic N-terminal B domain (residues 1-21), designated g32P-B, as well as its physiochemical and nucleic acid binding properties. G32P-B is readily purified from the soluble fraction of Escherichia coli BL21 (DE3) transformed with the plasmid pT7g32-B.wt which contains the g32P-B coding sequences under inducible transcriptional control of T7 RNA polymerase. Anion exchange, ssDNA-cellulose and phenyl-Sepharose chromatographies give rise to highly homogeneous g32P-B, free of contaminating nucleic acid. Recombinant g32P-B has the expected N-terminal primary structure and contains stoichiometric Zn(II). It also has the expected globular structure as shown by 1H NMR spectroscopy, hydrodynamic measurements, and the ability to selectively remove the carboxyl-terminal "A" domain to form the trypsin-resistant g32P-(A + B) DNA-binding core fragment. Quantitative ss nucleic acid binding experiments of g32P-B to poly(dT) (0.05 M NaCl, pH 8.1, 20 degrees C) show that all equilibrium binding isotherms can be fit with omega = 1 and Kobs = 5.2 (+/- 1.6) x 10(5) M-1, with a moderate electrostatic component to the binding free energy, delta log Kobs/delta log[NaCl] = -3.0 +/- 0.2. Under identical solution conditions, g32P-(A + B) derived from g32P-B binds to poly(dT) noncooperatively as expected, but with an approximately 80-fold higher apparent affinity, Kobs = 4.0 (+/- 2.0) x 10(7) M-1, and detectable enhanced salt sensitivity, delta log Kobs/delta log[NaCl] = -3.9 +/- 0.3. As the salt concentration is raised, the relative difference in Kobs between the g32P-(A + B) and g32P-B is gradually reduced such that extrapolation of the log-log plots to 1 M Na+ standard state gives similar Kobs within experimental error. Qualitatively similar observations are also found upon binding to the ribohomopolymer, poly(U).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Overexpression, purification, and characterization of recombinant T4 gene 32 protein22-301 (g32P-B). 219 20

Genomic and cDNA clones homologous to the RpII215 gene of Drosophila were isolated from Arabidopsis thaliana and assigned to a single copy gene encoding a transcript of 6.8 kb. Nucleotide sequence analysis of Arabidopsis genomic and cDNAs revealed a striking homology to yeast, Caenorhabditis, Drosophila and mouse genes encoding the largest subunit of RNA polymerase II. The Arabidopsis gene rpII215 contains 13 introns, 12 of which interrupt the coding sequence of a protein of 205 kDa. The position of the first intron is conserved between plant and animal genes, while an intron located in the 3' untranslated region of the rpII215 gene is unique to Arabidopsis. Common domains present in all known largest subunits of eucaryotic RNA polymerase II were identified in the predicted sequence of the Arabidopsis RpII215 protein. Both the order and the position of N-terminal Zn2+ finger and of DNA and alpha-amanitin binding motifs are conserved in Arabidopsis. The C-terminal region of the Arabidopsis protein contains 15 consensus and 26 variant YSPTSPS repeats (CTDs). Highly conserved structure among the various C-terminal domains suggests that the largest subunit of RNA polymerase II in plants may also interact with transcription factors and with protein kinases that control the cell cycle as in other organisms.
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PMID:Homologous domains of the largest subunit of eucaryotic RNA polymerase II are conserved in plants. 225 44


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