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)

In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase. Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro. Mutant analysis showed the sequence determinants of this dependence to lie in the region between the -10 box and the transcription start site. As with most promoters subject to stringent control, this portion of the hisR promoter is C-G-rich. Replacing a C/G bp with T/A at position -7 partially relieves the supercoiling response while changing the sequence between -5 and + 1 (-CCCCCG-) for -GTTAA- abolishes the response in vitro and in vivo. The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro. Studies in isoleucine-starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively. The data presented suggest that the hisR promoter's sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e. resistance to melting. We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix.
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PMID:The supercoiling sensitivity of a bacterial tRNA promoter parallels its responsiveness to stringent control. 955 Jul 33

We have isolated spontaneous rifampicin-resistant mutants from Escherichia coli that showed allele-specific suppression of the copy-number phenotype of ColE1 high-copy-number mutants in vivo. The key step in the regulatory circuitry of the initiation of ColE1 DNA replication is the formation of the persistent hybrid between the primer RNA and the DNA template around the replication origin. Three host-encoded enzymes, RNase H, DNA polymerase I, and RNA polymerase, are essential to the replication initiation in vitro. To decide whether the activity of RNA polymerase is involved directly in the formation of the persistent hybrid, we screened rifampicin-resistant colonies for suppressors of ColE1 copy-number mutants. Suppressor strain YY572 (rpoB572) changes the 572 residue of the beta subunit of RNA polymerase, encoded by the rpoB gene, from isoleucine to leucine. Another suppressor, YY513 (rpoB513), changes the 513 residue from glutamine to lysine. The other known rifampicin-resistant alleles located at residue 513, rpoB8 and rpoB101, did not show a significant suppression of the copy number of those ColE1 copy-number mutants as rpoB513. The suppression by rpoB513 on different ColE1 copy-number mutants showed allelic specificity. The possible roles of RNA polymerase in control of ColE1 copy number are discussed.
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PMID:Allele-specific suppression of ColE1 high-copy-number mutants by a rpoB mutation of Escherichia coli. 988 6

The sigma subunit of eubacterial RNA polymerase is essential for initiation of transcription at promoter sites. It directs recognition of DNA sequences by holoenzyme (alpha2betabeta'sigma) and facilitates subsequent steps in the initiation pathway. The primary sigma factor from Escherichia coli, sigma70, has four regions that are conserved among members of the sigma70 family. Previous work has shown that region 1.1 modulates DNA binding by regions 2 and 4 when sigma is separated from the core subunits, and is required for efficient progression through the later steps of initiation in the context of holoenzyme. In this report, we show that an amino acid substitution at position 53 in region 1.1, which converts isoleucine to alanine (I53A), creates a sigma factor that associates with the core subunits to form holoenzyme, but the holoenzyme is severely deficient for promoter binding. The I53A phenotype can be suppressed by truncation of five amino acids from the C-terminus of sigma70. We propose that the behavior of sigma70-I53A is a consequence of impaired ability to undergo a critical conformational change upon binding to the core subunits, which is needed to expose the DNA-binding domains and confer promoter recognition capability upon holoenzyme.
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PMID:A mutation in region 1.1 of sigma70 affects promoter DNA binding by Escherichia coli RNA polymerase holoenzyme. 992 30

Escherichia coli transcription factor sigma 54 contains motifs that resemble closely those used for RNA polymerase II in mammalian cells, including two hydrophobic heptad repeats, a very acidic region and a glutamine-rich region. Triple changes in hydrophobic or multiple changes in acidic residues in Region III are known to severely impair core-binding ability. To investigate whether all the changes in triple mutants are necessary for core binding, site-directed mutagenesis was performed to create single and double mutants in the leucine or isoleucine residues in the heptad repeat in Region III. Single mutants showed no discernible loss of function. Double mutants showed partial protection of the -12 promoter element of the glnAp2 promoter due to the partial loss of their ability to bind core RNA polymerase. These mutations were deleterious to the function of sigma 54, which retained only 30-40% of wild-type mRNA levels. However, double mutants retained nearly normal ability to form open complexes. Two triple mutants created during previous work lost most, if not all, of their ability to bind core RNA polymerase, to protect the -12 promoter element of the glnAp2 promoter and to open the transcription start site. The two triple mutants produced about 20% or less than 10% of the wild-type transcripts from the glnAp2 promoter. These results demonstrate that the hydrophobic heptad repeat in Region III is essential for core RNA polymerase binding. Progressive loss of hydrophobicity of the hydrophobic heptad repeat in Region III of sigma 54 resulted in a progressive loss of core-binding ability, leading to the loss of -12 promoter element recognition and mRNA production.
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PMID:The hydrophobic heptad repeat in Region III of Escherichia coli transcription factor sigma 54 is essential for core RNA polymerase binding. 1058 15

The reported crystal structures of plant and animal lipoxygenases (LOX) show that the nonheme iron in the catalytic domain is ligated by three histidines, the C-terminal isoleucine, and in certain structures also by a fifth iron ligand, an asparagine or histidine residue. Mouse 8-LOX and its homologues (e.g., human 15-LOX-2) are unique in having a serine in place of the usual Asn or His in this fifth position. To investigate the importance of the residue in mouse 8-LOX structure-function, the serine-558 was replaced by asparagine, histidine, or alanine using oligonucleotide-directed mutagenesis. Wild-type mouse 8-LOX and the mutant cDNAs were expressed in HeLa cells infected with vaccinia virus encoding T7 RNA polymerase and their relative lipoxygenase activities assessed by incubation with [14C]arachidonic acid or [14C]linoleic acid followed by HPLC analysis of the products. The Ser558Asn and Ser558His mutants had equivalent or greater activity than wild-type 8-LOX. They also exhibited some 15-LOX activity, indicating that small structural perturbations (in this case to a residue identical in mouse 8-LOX and its 15-LOX-2 homologues) can interchange the positional specificity of these closely related enzymes. Remarkably, the Ser558Ala mutant exhibited significant 8-LOX activity, indicating that this position is not an essential iron ligand in the enzyme. We conclude that mouse 8-LOX is catalytically competent with only four amino acid iron ligands, and that Ser-558 of the wild-type enzyme does not play an essential role in catalysis.
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PMID:Site-directed mutagenesis studies on a putative fifth iron ligand of mouse 8S-lipoxygenase: retention of catalytic activity on mutation of serine-558 to asparagine, histidine, or alanine. 1136 35

Escherichia coli glutaredoxin 2 (Grx2, encoded by grxB) differs greatly from the other two glutaredoxins in structure and catalytic properties. In a wild type strain, levels of Grx2 increased 3-fold in the stationary phase (up to 8 microg/mg). Guanosine-3',5'-tetraphoshate (ppGpp) and sigma(S), which regulate the transcription of genes in the stationary phase, dramatically affected the expression of Grx2. spoTrelA null mutants, lacking ppGpp, had very low levels of Grx2, while overproduction of full-length RelA or valine-induced starvation of isoleucine, both conditions elevating ppGpp levels, resulted in elevation of Grx2. Null mutants for the sigma(S)-specific protease ClpP, which have higher levels of sigma(S), exhibited a 3-fold Grx2 increase. sigma(S) in trans also increased the levels of Grx2. Therefore the stationary phase expression of Grx2 is determined by the sigma(S)-bound form of RNA polymerase in connection with ppGpp, while basal levels should be attributed to sigma(70)-RNA polymerase holoenzyme. Osmotic pressure and cAMP also affected the expression of Grx2, presumably via sigma(S). Furthermore, Grx2 levels were elevated in an oxyR(-) strain. In accordance with the role of Grx2 as a stationary phase protein, null mutants for grxB were shown to lyse under starvation conditions and exhibited a distorted morphology.
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PMID:Expression of Escherichia coli glutaredoxin 2 is mainly regulated by ppGpp and sigmaS. 1188 38

Region 2.1 of the sigma factor is once proposed to be involved in core binding, and certain bulky hydrophobic amino acids in region 2.1 are thought to make contact with the conserved isoleucine residues in the promoter -10 binding region on the same protein. To examine the roles of the contact between these two regions in sigma(A) structure and function, sigma(A )factor with L145A, I149A, or Y153A was created, and the effects of each substitution on the growth of Bacillus subtilis and on the structural and functional properties of sigma(A) were analyzed. Our data revealed that the growth potential of B. subtilis was significantly affected by each of the substitutions of sigma(A) at elevated temperature. The growth defect was most pronounced with the strain containing L145A-sigma(A); it possessed a low growth potential even at 37 degrees C. In parallel, changes in the structural stability and core-binding activity of sigma(A) and in the promoter-binding and transcription activities of sigma(A)-RNA polymerase were observed for each of the substitutions, with the most drastic effects exerted by L145A. Clearly, region 2.1 of sigma(A) has extra functions, such as the binding of RNA polymerase to promoter DNA, other than the known core-binding ability. Moreover, the multiple effects of each of the substitutions on sigma(A) demonstrate that the contacts between the hydrophobic amino acids in region 2.1 and those in the promoter -10 binding region are critical to the maintenance of the functional sigma(A) structure and that L145 in region 2.1 plays an important role in this respect.
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PMID:The importance of region 2.1 in sustaining the functional structure of the Bacillus subtilis sigma(A) factor. 1209 57

The Cyc8p/Tup1p complex mediates repression of diverse genes in Saccharomyces cerevisiae and is recruited by DNA binding proteins specific for the different sets of repressed genes. By screening the yeast deletion library, we identified Cyc8p as a coactivator for Gcn4p, a transcriptional activator of amino acid biosynthetic genes. Deletion of CYC8 confers sensitivity to an inhibitor of isoleucine/valine biosynthesis and impairs activation of Gcn4p-dependent reporters and authentic amino acid biosynthetic target genes. Deletion of TUP1 produces similar but less severe activation defects in vivo. Although expression of Gcn4p is unaffected by deletion of CYC8, chromatin immunoprecipitation assays reveal a strong defect in binding of Gcn4p at the target genes ARG1 and ARG4 in cyc8Delta cells and to a lesser extent in tup1Delta cells. The defects in Gcn4p binding and transcriptional activation in cyc8Delta cells cannot be overcome by Gcn4p overexpression but are partially suppressed in tup1Delta cells. The impairment of Gcn4p binding in cyc8Delta and tup1Delta cells is severe enough to reduce recruitment of SAGA, Srb mediator, TATA binding protein, and RNA polymerase II to the ARG1 and ARG4 promoters, accounting for impaired transcriptional activation of these genes in both mutants. Cyc8p and Tup1p are recruited to the ARG1 and ARG4 promoters, consistent with a direct role for this complex in stimulating Gcn4p occupancy of the upstream activation sequence (UAS). Interestingly, Gcn4p also stimulates binding of Cyc8p/Tup1p at the 3' ends of these genes, raising the possibility that Cyc8p/Tup1p influences transcription elongation. Our findings reveal a novel coactivator function for Cyc8p/Tup1p at the level of activator binding and suggest that Gcn4p may enhance its own binding to the UAS by recruiting Cyc8p/Tup1p.
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PMID:Activator Gcn4p and Cyc8p/Tup1p are interdependent for promoter occupancy at ARG1 in vivo. 1631 36

The reverse-transcriptase inhibitor lamivudine (Zeffix, GlaxoSmithKline) is often used to treat chronic infection with hepatitis B virus (HBV) until resistance develops. Treatment may then be switched to the reverse-transcriptase inhibitor adefovir (Hepsera, Gilead), which has a lower frequency of resistance. Here, we describe three cases of primary adefovir resistance that were sensitive to tenofovir (Viread, Gilead). All three cases involved a rare HBV variant with a valine at position 233 of the reverse-transcriptase domain instead of isoleucine (rtI233V), as in the wild-type virus. This HBV variant also displayed resistance to adefovir and sensitivity to tenofovir in vitro.
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PMID:Variant of hepatitis B virus with primary resistance to adefovir. 1685 78

Protein-protein interactions are crucial to biological functions. Consequently, designing drugs to control protein-protein interactions is receiving increasing attention. Protein structures can associate in different ways. Analysis of the structures of protein-protein complexes using amino acid sequence order-independent multiple structural comparison algorithms, led us to conclude that the amino acids Trp, Met, and Phe are important for protein-protein interactions. Hence, in principle, drug design targeting the Trp/Met/Phe should modulate protein functions effectively. Several clusters of the Trp/Met/Phe residues are involved in the p53 protein-protein interactions. The best example in this regard is the Phe19/Trp23 of p53, which binds to transcriptional factors and to the MDM2 protein. In the HIV related proteins, the Trp/Met/Phe residues have roles in the dimerization of the transcriptase (p51/p66) and in cell-fusion processes, including the gp120-CD4 interaction and the gp41 six-helix bundle formation. Trp/Met/Phe residues are preferred in 'normal' functional protein-protein interactions and they also appear to be exploited in amyloid formation, especially the phenylalanine. Comparison of binding propensity and amyloid formation preference reveals that apart from Lysine, Isoleucine is the least structurally conserved in protein binding sites and has a high propensity in sequences forming amyloids. Thus, this may suggest that nature tends to avoid Ile conservation in protein-protein interaction to avoid amyloid formation. In this regards, Trp/Met/Phe as well as Ile may be targeted to modulate protein-protein interaction.
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PMID:Trp/Met/Phe hot spots in protein-protein interactions: potential targets in drug design. 1750 33


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