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)

Four amatoxin-binding proteins with KD values in the nanomolar range, 3 monoclonal antibodies and RNA polymerase II, were studied with respect to their affinities to 24 alpha-amanitin derivatives with modified side chains. From KD values we estimated the amounts of binding energy that single side chains of the amatoxins contribute to complex formation. Ile6, previously identified by X-ray analysis to be part of a beta-turn (Kostansek EC, Lipscomb WN, Yocum RR, Thiessen WE, 1978, Biochemistry 17:3790-3795) proved to be of outstanding importance in all complexes. Replacement of the isoleucine with alanine reduced the affinity to all binding proteins to < 1%, suggesting a strong hydrophobic interaction. A strong effect was also seen when Gly5 was replaced with alanine, suggesting that the absence of a side chain in proximity to the beta-turn is likewise important. In addition to the beta-turn, each of the proteins showed at least 2 other points of strong contact formed by hydrogen bonds. Donors are the indole NH of 6'-hydroxy-Trp4 and OH of hydroxy-Pro2 and dihydroxy-Ile3. All the antibodies, but not RNA polymerase II, recognized the indole nucleus of 6'-hydroxy-Trp4. The geometric arrangement of the 4 strongest contact points suggests that the amatoxin binding site is different in each of the 4 proteins, except for the 2 antibodies raised in the same animal. Here, most of the contact points were identical but differed in strength of interaction. The method of structural analysis presented in this study is useful for identifying contact sites in complexes of proteins with peptides of rigid conformation. Furthermore, the method complements X-ray data by providing information on the amount of binding energy contributed by single structural elements.
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PMID:A beta-turn in alpha-amanitin is the most important structural feature for binding to RNA polymerase II and three monoclonal antibodies. 806 5

It has been demonstrated that the amino acids Asp537, Asp812, Lys631, His811 and Tyr639 are involved in bacteriophage T7 RNA polymerase catalysis. In the present paper, we report kinetic, spectroscopic and calorimetric characterization of the wild-type and mutant T7 RNA polymerases generated at these five loci (D537N, E; K631M, R; Y639F, S, A, W; H811Q, A; D812N, E). The wild-type enzyme has a substantial amount of secondary structure as determined by CD analysis (alpha-helix, 43%; beta-sheet, 14%; beta-turn, 25%; unordered, 18%). The CD spectra of 12 mutants at five loci are very similar to that of the wild-type, except for the mutant Y639W. Within experimental error, the thermal transition temperatures measured by CD and DSC as well as the lambda max values of the fluorescence spectra were the same for the wild-type and all of the mutants. Therefore, the overall folding and stability of the mutant enzymes are very similar to those of the wild-type enzyme, although small local conformational changes cannot be excluded. For the synthesis of the pentamer pppGGACU, the mutants D537E and D812E showed an approximately two- to threefold decrease in (kcat)app and an approximately two- to threefold increase in (Km)app, relative to the wild-type, in contrast to the mutants D537N and D812N which exhibited no detectable activity. The mutant K631R showed a sevenfold reduction in (kcat)app and a two- to threefold increase in (Km)app, supporting our earlier observation with the mutant K631M that Lys631 may be involved in phosphodiester bond formation. The mutant Y639S can synthesize the trimer GGA with an approximately 50-fold decrease in (kcat)app and a tenfold increase in (Km)app, relative to the wild-type, underlining the importance of the phenyl ring of Tyr639. The mutant H811A, in which the side-chain at position 811 is incapable of forming a hydrogen bond, can synthesize the trimer GGA with an approximately tenfold decrease in (kcat)app and an approximately 35-fold increase in (Km)app. Thus, either the hydrogen-bonding capacity of this residue is non-essential or some other group can functionally substitute for the His811 side-chain. The wild-type enzyme showed significant effects of the base position in the sequence on the apparent binding constants for the NTPs. The kinetics of GpG-primed trimer, tetramer and pentamer synthesis on three 22 bp templates were investigated for the wild-type and mutant enzymes with measurable activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Bacteriophage T7 RNA polymerase and its active-site mutants. Kinetic, spectroscopic and calorimetric characterization. 813 19

The kinetically component open complexes formed at prokaryotic and eukaryotic transcription start sites are efficiently nicked by the chemical nuclease activity of the 2:1 1,10-phenanthroline-copper(I) complex [(OP)2Cu+] and hydrogen peroxide. This reaction specificity has been attributed to the creation of a binding site(s) for redox-active tetrahedral (OP)2Cu+ when RNA polymerase form productive complexes with promoters. This proposal has been confirmed for the Escherichia coli lac UV-5 promoter by the demonstration that the 2:1 2,9-dimethyl-1,10-phenanthroline-copper(I) complex [(Me2OP)2Cu+], a redox-inactive isostere of (OP)2-Cu+, protects the transcription start site from scission by the chemical nuclease activity. (Me2OP)2Cu+ is also an effective inhibitor of transcription. The inhibition of transcription and the protection from scission of the open complex by (OP)2Cu+ exhibit the same dependence on the concentration of (Me2OP)2Cu+. This redox- and exchange-stable species is a previously undescribed transcription inhibitor that binds to a site generated by the interaction of RNA polymerase with the promoter. Unlike the intercalating agent proflavine, which is also an effective transcription inhibitor, it does not displace the enzyme from the promoter. The ability of (Me2OP)2Cu+ to inhibit transcription may be partially responsible for its potent cytotoxicity.
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PMID:A transcription inhibitor specific for unwound DNA in RNA polymerase-promoter open complexes. 836 75

In vitro specific transcription by the Rickettsia prowazekii RNA polymerase was investigated. The purified rickettsial RNA polymerase, in striking contrast to that of Escherichia coli, could specifically transcribe two R. prowazekii genes (ATP/ADP translocase and citrate synthase genes) and one E. coli gene (RNA-I) on negative supercoiled plasmids but not the same genes on linear plasmids. Following the specific binding of the rickettsial RNA polymerase to the translocase gene promoter on a linear plasmid, there was no detectable open complex formation. Both the E. coli and the R. prowazekii RNA polymerases worked well when poly(dA-dT).poly(dA-dT) or poly(dI-dC).poly-(dI-dC) was used as template for generalized transcription. However, the rickettsial RNA polymerase, in contrast to the E. coli enzyme, had little activity on poly(dG-dC).poly(dG-dC), a template with a larger number of hydrogen bonds. These data indicate that the rickettsial RNA polymerase is weak, at least relative to E. coli, in the function required for the opening of DNA duplex. It appears that this operation in R. prowazekii is aided by the negative supercoiling and the high 72% AT composition of the rickettsial genome.
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PMID:Characterization of the DNA-melting function of the Rickettsia prowazekii RNA polymerase. 844 Jun 83

In Rhizobium meliloti, transcription of the key nitrogen-fixation regulatory genes nifA and fixK is induced in response to microaerobiosis through the action of the FixL and FixJ proteins. These two proteins are sensor and regulator homologues, respectively, of a large family of bacterial two-component systems involved in sensing and responding to environmental changes. A soluble, truncated form of the membrane protein FixL, FixL*, has been shown to be a hemoprotein that phosphorylates and dephosphorylates FixJ in response to oxygen tension. Here we use an in vitro transcription system to prove that FixJ is a transcriptional activator of both nifA and fixK and that phosphorylation of FixJ markedly increases its activity. Phosphorylation was achieved either by preincubating FixJ with FixL* and ATP or by exposing FixJ to the inorganic phospho donor ammonium hydrogen phosphoramidate. Both FixJ and FixJ-phosphate formed heparin-resistant complexes under the assay conditions used. Lastly, we were able to show that anaerobiosis, in the presence of FixL* and ATP, greatly stimulates FixJ activity at the nifA promoter with either Escherichia coli or R. meliloti RNA polymerase. This use of atmospheric oxygen to control nifA transcription in vitro represents a reconstitution of a bacterial two-component signal transduction system in its entirety, from effector to ultimate target, by the use of purified components.
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PMID:Oxygen regulation of nifA transcription in vitro. 847 99

The DNA-dependent RNA polymerase from bacteriophage T7 is highly specific for a 17 base promoter sequence. Interactions between T7 RNA polymerase and its promoter DNA have been probed using modified oligonucleotides and a steady-state kinetic assay. The incorporation of deoxyuridine in place of thymidine at individual sites in the promoter sequence results in the replacement of an exocyclic methyl group by hydrogen (effectively removing the thymine methyl). This substitution has been placed individually at each of the thymines in the T7 consensus promoter. Many of these substitutions do not affect binding or catalysis; however, the thymine methyl group at position-6 is critical to recognition. The kinetic parameter Km increases approximately 10-fold while kcat is only slightly affected, suggesting that this thymine methyl is critical to binding specificity, but not to the kinetics of initiation. Two methyl groups near the start site on the template strand (at positions -1 and -3) also contribute to promoter specificity, while nearby methyl groups on the nontemplate strand do not. The implications of these results are discussed with respect to recent models for promoter binding.
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PMID:Tests of a model for promoter recognition by T7 RNA polymerase: thymine methyl group contacts. 847 55

The heterologous expression and spectroscopic characterization of the [2Fe-2S] ferredoxin from the sexually transmitted human parasite Trichomonas vaginalis is described. Using oligonucleotide primers based on the deduced DNA sequence, the gene encoding the ferredoxin was amplified by polymerase chain reaction and cloned into a T7 RNA polymerase expression vector. Expression of the gene in Escherichia coli host HMS174(DE3) resulted in the high level production of the protein with the correctly assembled iron-sulfur cluster. The absorption, circular dichroism, resonance Raman, and EPR spectra of the recombinant protein revealed many differences from those of other [2Fe-2S] ferredoxins. The redox potential of the protein (-347 mV versus normal hydrogen electrode) was also determined. Whereas the amino acid sequence of T. vaginalis ferredoxin showed greatest homology to the [2Fe-2S] ferredoxins found in bacteria and vertebrate mitochondria which function in cytochrome P450 oxidation pathways, the spectroscopic properties showed substantial dissimilarity. Differences in the biophysical properties and function of T. vaginalis ferredoxin are proposed to result from the characteristic amino acid sequence of the parasite protein near the cysteine residues that ligate the valence-localized Fe(III) site of the reduced cluster.
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PMID:Expression and spectroscopic characterization of the hydrogenosomal [2Fe-2S] ferredoxin from the protozoan Trichomonas vaginalis. 866 34

We report the use of novel non-polar nucleoside analogues as terminators of enzymatic RNA and DNA synthesis. Standard 'runoff' RNA synthesis by T7 RNA polymerase gives RNA products which have ragged ends as a result of transcription which often extends beyond the end of the template DNA strand. Similarly, the Klenow fragment of Escherichia coli DNA polymerase I tends to run past the end of the template strand during DNA synthesis. We report here that certain non-hydrogen-bonding nucleoside analogues, when placed at the downstream 5'-end of a template DNA strand, cause the polymerases to stop more abruptly at the last coding nucleotide. This results in a considerably more homogeneous oligonucleotide being produced. Three novel nucleosides are tested as potential terminators: 4-methylindole beta-deoxynucleoside (M), 1-naphthyl alpha-deoxynucleoside (N) and 1-pyrenyl alpha-deoxynucleoside (P). Comparison is made to an abasic nucleoside (phi) and to unterminated synthesis. Of these, M is found to be the most efficient at terminating transcription, and both P and M are highly effective at terminating DNA synthesis. It is also found that the ability of a nucleoside to stall synthesis when it is internally placed in the template strand is not necessarily a good predictor of terminating ability at the end of a template. Such terminator nucleosides may be useful in the preparative enzymatic synthesis of RNA and DNA, rendering purification simpler and lowering the cost of synthesis by preventing the uptake of potentially costly nucleotides into unwanted products.
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PMID:Non-hydrogen bonding 'terminator' nucleosides increase the 3'-end homogeneity of enzymatic RNA and DNA synthesis. 866 34

A 16-base pair oligo(purine)-oligo(pyrimidine) sequence present in the coding region of two HIV 1 proviral genes (pol and nef) was chosen as a target for triplex-forming oligonucleotides in in vitro transcription assays. Inhibition of transcription elongation was observed with triplex-forming oligonucleotide-acridine conjugates (Acr-15-TCG:5'-Acr-T4CT4G6-3' and Acr-9-TC:5'-Acr-T4CT4-3' where C is 5-methylcytosine) under conditions where the unsubstituted oligomers did not exhibit any inhibitory effect. Both SP6 bacteriophage RNA polymerase and eukaryotic RNA polymerase II were physically blocked by such a triplex barrier. The polymerase arrest is caused by the triple-helical complex involving the hydrogen-bonded oligonucleotide stabilized by the intercalated moiety and not solely by the acridine molecule specifically intercalated at the duplex-triplex junction. The stability of the triple-helical complex formed by the 15-mer containing thymines, cytosine, and guanines (15-TCG) and involving the formation of six contiguous C.GxG base triplets was strongly enhanced in the presence of a benzopyridoindole derivative (BePI), which intercalates in triplex structures. This improvement of the binding affinity led to an increased inhibition of transcription elongation. The present results demonstrate the necessity to use triplex-forming oligonucleotides with high binding affinity and a long residence time on their double-stranded target to efficiently inhibit transcription elongation. These data provide a rational basis for the optimization and the development of triplex-forming oligonucleotides as transcriptional blockers, even when they are targeted to the transcribed portion of a gene, downstream of the transcription initiation site.
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PMID:Specific inhibition of in vitro transcription elongation by triplex-forming oligonucleotide-intercalator conjugates targeted to HIV proviral DNA. 875 10

clpC of Bacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (PA and PB) were mapped upstream of the first gene. PA resembles promoters recognized by the vegetative RNA polymerase E sigma A. The other promoter (PB) was shown to be dependent on sigma B, the general stress sigma factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a sigma B-dependent manner. This is the first evidence that sigma B in B. subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by sigma 32 in Escherichia coli, indicating a new function of sigma B-dependent general stress proteins. PB deviated from the consensus sequence of sigma B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the sigma B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the sigma A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream sigma A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.
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PMID:Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon. 879 70


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