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

The Bacillus subtilis sigA gene encodes the primary sigma factor of RNA polymerase and is essential for cell growth. We have mutated conserved region 2.3 of the sigma A protein to substitute each of seven aromatic amino acids with alanine. Several of these aromatic amino acids are proposed to form a melting motif which facilitates the strand separation step of initiation. Holoenzymes containing mutant sigma factors recognize promoters, but some are defective for DNA melting in vitro. We have studied the ability of each mutant sigma factor to support cell growth by gene replacement and complementation. The two region 2.3 mutants least impaired in promoter melting in vitro (Y180A and Y184A) support cell growth in single copy, although the Y184A allele imparts a slow-growth phenotype at low temperatures. A strain expressing only the Y189A variant of the sigma A protein, known to be defective in DNA melting in vitro, grows very slowly and is altered in its pattern of protein synthesis. Only the wild-type and Y180A sigma A proteins efficiently complement a temperature-sensitive allele of sigA. Overexpression of three of the sigma A proteins defective for promoter melting in vitro (Y189A, W192A, and W193A) leads to a decrease in RNA synthesis and cell death. These results indicate that mutations which specifically impair DNA melting in vitro also impair sigma function in vivo and therefore support the hypothesis that sigma plays an essential role in both DNA melting and promoter recognition.
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PMID:Genetic and physiological studies of Bacillus subtilis sigma A mutants defective in promoter melting. 807 Nov 96

The vaccinia virus D6R open reading frame encodes the small subunit of the heterodimeric vaccinia virus early transcription factor (VETF) that activates transcription of early genes in vitro. VETF binds early gene promoters and has a DNA-dependent ATPase activity that is essential for activation of transcription. To examine the relationship between the structure and function of VETF, we have localized the mutations in two temperature-sensitive viruses whose lesions previously were mapped to the D6R gene. For both mutants, a single G-to-A nucleotide change that would alter protein coding potential was identified. In mutant E93, the codon for alanine 25 was changed to that of threonine, and in mutant S4 the codon for valine 278 was replaced with that for methionine. The molecular phenotype of each mutant was assessed by expressing mutant transcription factors in HeLa cells by using a vaccinia virus-T7 system and characterizing the proteins' activities in vitro. The A25T mutant activated transcription to a lesser extent than wild-type VETF, and the V278M mutant had no demonstrable transcription factor activity. Both mutant proteins were shown to be defective for promoter binding, accounting for their impairment in transcription activation. The functional defects for both mutants were observed at permissive as well as nonpermissive temperatures. The mutant proteins retained ATPase activity but required higher DNA concentrations to activate the ATPase. These results indicate that the small subunit of VETF is essential for its promoter binding activity and likely contacts the promoter DNA. Immunoblotting experiments showed that the virion particles from the two mutant viruses contained about half the VETF of wild-type virus, suggesting that promoter binding may contribute to packaging of VETF into the virion particle. RNA polymerase, mRNA capping enzyme, and nucleoside triphosphate phosphohydrolase I were found at similarly reduced levels in the virion, indicating that packaging of some virion core enzymes may be interdependent.
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PMID:Temperature-sensitive mutations in the gene encoding the small subunit of the vaccinia virus early transcription factor impair promoter binding, transcription activation, and packaging of multiple virion components. 813 39

Three synthetic peptides, pyro-Glu-Ala-Gly-Glu-Ser-Glu-Asp (Pep A), pyro-Glu-Ala-Gly-Glu-Glu-Glu-Ser-Asn (Pep B), and pyro-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn (Pep C), bear sequences possibly belonging to components of a naturally occurring family of strongly related small acidic chromatin peptides involved in regulation of gene expression. In a crude nuclear fraction and in purified nuclei from PC-12 cells, Pep A and Pep B activate RNA synthesis, specifically acting on the RNA polymerase II transcription system. On the other hand, Pep C shows an inhibitory effect on RNA synthesis in purified nuclei but an activation in the crude nuclear fraction. Control experiments show that the serum thymic factor does not affect RNA synthesis in the crude nuclear fraction or in purified nuclei. A possible regulation by peptide phosphorylation via casein kinase II (more active in purified nuclei than in the crude nuclear fraction) is discussed.
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PMID:Possible specific activation of RNA synthesis in PC-12 cell isolated nuclei by small acidic peptides. 823 75

The NTRC protein of enteric bacteria is an enhancer-binding protein that activates transcription by the sigma 54-holoenzyme form of RNA polymerase under nitrogen-limiting conditions. In vitro NTRC must be phosphorylated to catalyze ATP hydrolysis and activate transcription. The site of phosphorylation of NTRC from Salmonella typhimurium is Aspartate 54, which lies in the amino-terminal regulatory domain of the protein. We used site-directed mutagenesis to make "conservative" substitutions at residue 54 to alanine, asparagine, and glutamate, and examined the properties of the mutant NTRC proteins in vitro and in vivo. In vitro none of them was detectably phosphorylated, as expected if D54 is, in fact, the sole site of phosphorylation. D54A and D54N did not activate transcription of glnA but, interestingly, D54E activated constitutively. Activation by D54E was partial compared to that by phosphorylated wild-type NTRC. Combining D54A or D54N with S160F, a change in the central domain of NTRC that partially bypasses the requirement for phosphorylation, yielded doubly mutant proteins that were as active as a form carrying S160F alone, indicating that the changes in D54 did not adversely affect the function of the remainder of NTRC. Combining D54E with S160F increased the levels of constitutive ATPase activity and transcriptional activation above those of mutant NTRC proteins carrying either single change alone. We conclude that phosphorylation of aspartate 54 is required to activate NTRC and postulate that the D54E mutation mimics phosphorylation, thereby allowing NTRC to hydrolyze ATP and activate transcription. Phenotypes of mutant strains encoding NTRC proteins with substitutions at D54 indicated that phosphorylation of NTRC at position 54 was necessary for normal growth in the absence of glutamine and that such phosphorylation occurred to some extent even in the absence of NTRB.
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PMID:Glutamate at the site of phosphorylation of nitrogen-regulatory protein NTRC mimics aspartyl-phosphate and activates the protein. 833 71

The use of synthetic tRNA for in vitro protein engineering was tested in a coupled transcription/translation system prepared from Escherichia coli. DNA sequences similar to the natural tRNA(Ala/UGC) gene from E. coli but with different anticodons were synthesized in vitro, cloned into a DNA plasmid, and then transcribed in vitro with T7 RNA polymerase. The UGC alanine anticodon was changed to CUA corresponding to the UAG stop codon, CCU corresponding to the rarely used AGG arginine codon, and two four-nucleotide anticodons used to suppress stop codons. Bacterial dihydrofolate reductase was the test protein. Its cloned coding sequence was mutagenized at the GUG codon for valine-75 to correspond to the anticodons of the tRNA constructs, and then the plasmids were used to direct the synthesis of dihydrofolate reductase in the coupled transcription/translation system containing the corresponding synthetic tRNA. The results indicate that all four synthetic tRNAs were functionally active in the synthesis of full-length, enzymatically active dihydrofolate reductase protein.
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PMID:In vitro protein engineering using synthetic tRNA(Ala) with different anticodons. 834 99

The role of Cys-138 in the catalysis of the skeletal muscle 6-phosphofructo-2-kinase reaction was investigated by mutating this residue to serine, glutamine and alanine, expressing the mutants in E. coli with a T7 RNA polymerase-based expression system, and analyzing their kinetic properties. The Cys138Ala mutant had greatly diminished activity, while the Cys138Ser and Cys138Gln mutants had maximal velocities 2-3 fold higher than the wild-type enzyme. It was concluded that Cys-138 does not act as a base catalyst in the kinase reaction, but that it plays a significant structural role in the enzyme's active site.
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PMID:Lack of evidence for a role of Cys-138 as a base catalyst in the skeletal muscle 6-phosphofructo-2-kinase reaction. 836 5

A cDNA encoding human liver fructose-1,6-bisphosphatase was isolated from a lambda gt11 library by screening with a rat liver fructose-1,6-bisphosphatase cDNA. The cDNA (1421 base pairs) contains an open reading frame encoding 337 amino acids, corresponding to a protein with an estimated molecular weight of 36,697. Its primary sequence is highly homologous to that of the pig kidney and rat liver enzymes. The human liver cDNA was used to construct a T7 RNA polymerase-transcribed expression vector, and the enzyme was expressed in Escherichia coli BL21 (DE3). Approximately 50% of the expressed human fructose-1,6-bisphosphatase was soluble and enzymatically active, and the enzyme was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and substrate/AMP elution from carboxymethyl-Sephadex. Expressed human liver fructose-1,6-bisphosphatase had a specific activity (9.8 mumol/min/mg of protein) that was half that of the rat liver enzyme, but had an identical Km for substrate. However, the human enzyme was more sensitive to inhibition by fructose-2,6-bisphosphate (Ki = 0.3 microM) and AMP (Ki = 12 microM) than the rat liver form (fructose 2,6-P2, Ki = 4 microM; AMP, Ki = 40 microM). Crystallographic analyses have suggested that Asp-118 and Asp-121 are catalytic residues located in a negatively charged pocket that binds divalent metal cations. These residues were mutated to alanine, and the E. coli-expressed mutant enzymes were purified to homogeneity. The Asp-118-->Ala and Asp-121-->Ala mutants had 1/5000 and 1/20,000 lower Kcat values than the wild-type enzyme, respectively, consistent with their critical role in fructose-1,6-bisphosphatase catalysis.
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PMID:Isolation of a human liver fructose-1,6-bisphosphatase cDNA and expression of the protein in Escherichia coli. Role of ASP-118 and ASP-121 in catalysis. 838 95

Host cell RNA polymerase II-mediated transcription is inhibited by poliovirus infection. We have shown previously that the human TATA-binding protein (TBP), a general transcription factor required for transcription of all RNA polymerase II genes, is directly cleaved both in vitro and in vivo by the virus-coded protease 3CPro. 3CPro specifically cleaves glutamine-glycine bonds in the viral polyprotein. Cellular transcription factor TBP contains three glutamine-glycine sites, at amino acids 12, 18, and 108. By using site-directed mutagenesis, we determined that the glutamine-glycine bond at amino acid 18, but not that at amino acid 12 or 108, is cleaved by the viral protease. Both the glutamine and the glycine appear to be important for the cleavage. Further mutations around the glutamine-glycine site at position 18 suggest that determinants other than the glutamine-glycine bond in TBP are also required for 3CPro-induced cleavage. An alanine at position P4 and a proline at position P2, proximal to the scissile glutamine-glycine pair, appear to be important for 3CPro-mediated cleavage of TBP. Our results suggest that the cleavage specificity of 3CPro for a cellular transcription factor is very similar to its mode of cleavage of viral polyproteins.
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PMID:Identification of the cleavage site and determinants required for poliovirus 3CPro-catalyzed cleavage of human TATA-binding transcription factor TBP. 838 2

Mutant a and alpha yeast cells were created with histone H3 containing cysteine in place of alanine 110. Because transcriptionally active nucleosomes "unfold" to reveal the histone H3-thiol groups at the center of the core, the active nucleosomes of the mutant strain can be isolated by mercury-affinity chromatography. We compared the unbound and mercury-bound nucleosomes of haploid H3-mutant strains expressing either the MAT alpha or the MATa mating-type locus. In a MAT alpha strain, the Hg-bound nucleosomes are enriched in MAT alpha DNA but lack the DNA of the transcriptionally silent HMRa mating-type locus. Conversely, in a MATa strain, the Hg-bound nucleosomes are enriched in MATa DNA sequences but deficient in HML alpha DNA. When the SIR3 gene, known to be required for silencing of the repressed mating-type loci, is mutated in the MAT alpha strain, transcription of the HMRa ensues, and its nucleosomes, as well as those of the MAT alpha locus, are retained by the organomercurial column. It follows that derepression of the silent mating-type locus, caused by the sir3 null mutation, is accompanied by an unfolding of its nucleosomes to reveal the histone H3 SH groups at their centers. Nucleosomes of the pheromone-encoding gene MFA2, a gene that is expressed in MATa cells but not in MAT alpha cells, are bound to the organomercurial column when isolated from MATa cells but not from MAT alpha cells. Thus, there is a good correlation between nucleosome unfolding and the renewed transcriptional activity at mating-type loci, and at MFA2, which had been silenced for prolonged periods. A close temporal correlation between nucleosome refolding and the cessation of transcription is not always observed in yeast, however, in contrast to observations in mammalian cells. For example, nucleosomes of the GAL1 gene are maintained in a "poised" or "primed" thiol-reactive state even when the gene is not being transcribed (Chen, T. A., Smith, M. M., Le, S., Sternglanz, R., and Allfrey, V. G. (1991) J. Biol. Chem. 266, 6489-6498). It follows that the unfolding of the nucleosome cores of the yeast H3 mutant is regulated by factors that are not temporally linked to the recruitment or traverse of the RNA polymerase complex, but which may determine the rate at which different domains of chromatin adapt to the need for transcription of the associated DNA sequences.
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PMID:Nucleosome structural changes during derepression of silent mating-type loci in yeast. 841 18


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