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)

Members of the protein family called ATPases associated with various cellular activities (AAA(+)) play a crucial role in transforming chemical energy into biological events. AAA(+) proteins are complex molecular machines and typically form ring-shaped oligomeric complexes that are crucial for ATPase activity and mechanism of action. The Escherichia coli transcription activator phage shock protein F (PspF) is an AAA(+) mechanochemical enzyme that functions to sense and relay the energy derived from nucleoside triphosphate hydrolysis to catalyze transcription by the sigma(54)-RNA polymerase. Closed promoter complexes formed by the sigma(54)-RNA polymerase are substrates for the action of PspF. By using a protein fragmentation approach, we identify here at least one sigma(54)-binding surface in the PspF AAA(+) domain. Results suggest that ATP hydrolysis by PspF is coupled to the exposure of at least one sigma(54)-binding surface. This nucleotide hydrolysis-dependent presentation of a substrate binding surface can explain why complexes that form between sigma(54) and PspF are transient and could be part of a mechanism used generally by other AAA(+) proteins to regulate activity.
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PMID:The ATP hydrolyzing transcription activator phage shock protein F of Escherichia coli: identifying a surface that binds sigma 54. 1260 Nov 52

ATP-dependent chromatin remodeling factors have been implicated in nuclear processes involving DNA. Here we report partial purification and characterization of an ATP-dependent chromatin remodeling activity from chicken liver. Nuclear extract from chicken liver was fractionated chromatographically to enrich proteins immunoreacting to antibodies against components of human SWI/SNF, namely BRG1, BAF170, BAF155, and BAF57. Immunoreactivity to these antibodies elutes with a mass of about 2MDa on Sepharose CL-6B gel filtration, suggesting that they constitute a SWI/SNF-like complex (SLC). The SLC displays three chromatin-remodeling activities, viz. nucleosome disruption, octamer transfer, and nucleosome sliding (octamer transfer in cis). We further show that components of SLC, as revealed by immunoreactivity to the above antibodies, display a dynamic nucleocytoplasmic distribution and colocalize with RNA polymerase II in the liver nuclei. This report contributes to the understanding of phylogenetic generality of chromatin remodeling factors in eukaryotes.
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PMID:A SWI/SNF-like factor from chicken liver that disrupts nucleosomes and transfers histone octamers in cis and trans. 1274 51

The stringent starvation protein A (SspA), an Escherichia coli RNA polymerase (RNAP)-associated protein, has been reported to be essential for lytic growth of bacteriophage P1. Unlike P1 early promoters, P1 late promoters are not recognized by RNAP alone. A phage-encoded early protein, Lpa (late promoter activator protein, formerly called gp10), has been shown to be required for P1 late transcription in vivo. Here, we demonstrate that SspA is a transcription activator for P1 late genes. Our results indicated that Lpa is not limiting in an sspA mutant. However, the transcription of P1 late genes was deficient in an sspA mutant in vivo. We demonstrated that SspA/Lpa are required for transcription activation of the P1 late promoter Ps in vitro. In addition, SspA and Lpa were shown to facilitate the binding of RNAP to Ps late promoter DNA. Activation of late transcription by SspA/Lpa was dependent on holoenzyme containing sigma70 but not sigmaS, indicating that the two activators discriminate between the two forms of the holoenzyme. Furthermore, P1 early gene expression was downregulated in the wild-type background, whereas it persisted in the sspA mutant background, indicating that SspA/Lpa mediate the transcriptional switch from the early to the late genes during P1 lytic growth. Thus, this work provides the first evidence for a function of the E. coli RNAP-associated protein SspA.
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PMID:Escherichia coli SspA is a transcription activator for bacteriophage P1 late genes. 1279 Nov 43

The cyclic AMP receptor protein (CRP) acts as a transcription activator at many promoters of Escherichia coli. We have examined the kinetics of open complex formation at the lacP1 promoter using tryptophan fluorescence of RNA polymerase and DNA fragments with 2-aminopurine substituted at specific positions. Apart from the closed complex formation and promoter clearance, we were able to detect three steps. The first step after the closed complex formation leads to a rapid increase of 2-aminopurine fluorescence. This was followed by another rapid step in which quenching of tryptophan fluorescence of RNA polymerase was observed. The slowest step detected by 2-aminopurine fluorescence increase is assigned to the final open complex formation. We have found that CRP not only enhances RNA polymerase binding at the promoter, but also enhances the slowest isomerization step by about 2-fold. Furthermore, potassium permanganate probing shows that the conformation of the open complex in the presence of CRP appears qualitatively and quantitatively different from that in the absence of CRP, suggesting that contact with RNA polymerase is maintained throughout the transcription initiation.
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PMID:Kinetics of transcription initiation at lacP1. Multiple roles of cyclic AMP receptor protein. 1288 19

Proteins that belong to the AAA (ATPases associated with various cellular activities) superfamily of mechanochemical enzymes are versatile and control a wide array of cellular functions. Many AAA proteins share the common property of self-association into oligomeric structures and use nucleotide binding and hydrolysis to regulate their biological output. The Escherichia coli transcription activator PspF (phage shock protein F) is a member of the sigma54-dependent transcriptional activators that belong to the AAA protein family. Nucleotide interactions condition the functional state of PspF, enabling it to self-associate and interact with its target, the sigma54-RNAP (RNA polymerase) closed complex. The self-association determinants within the AAA domain of sigma54-dependent activators remain poorly characterized. In the present study, we have used a fragment of the AAA domain of PspF as a probe to study the nucleotide-conditioned self-association of PspF. Results show that the PspF fragment acts in trans to inhibit specifically self-association of PspF. The PspF fragment prevented efficient binding of nucleotides to PspF, consistent with the observation that the site for nucleotide interactions within an oligomer of AAA proteins is created between two protomers. Using proximity-based footprinting and cross-linking techniques, we demonstrate that the sequences represented in this fragment are close to one protomer-protomer interface within a PspF oligomer. As the sequences represented in this PspF fragment also contain a highly conserved motif that interacts with the sigma54-RNAP closed complex, we suggest that PspF may be organized to link nucleotide interactions and self-association to sigma54-RNAP binding and transcription activation.
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PMID:Sigma54-dependent transcription activator phage shock protein F of Escherichia coli: a fragmentation approach to identify sequences that contribute to self-association. 1465

Transcription-coupled repair (TCR) and global genomic repair (GGR) of UV-induced cyclobutane pyrimidine dimers were investigated in the yeast GAL1-10 genes. Both Rpb9- and Rad26-mediated TCR are confined to the transcribed strands, initiating at upstream sites approximately 100 nucleotides from the upstream activating sequence shared by the two genes. However, TCR initiation sites do not correlate with either transcription start sites or TATA boxes. Rad16-mediated GGR tightly correlates with nucleosome positioning when the genes are repressed and are slow in the nucleosome core and fast in linker DNA. Induction of transcription enhanced GGR in nucleosome core DNA, especially in the nucleosomes around and upstream of the transcription start sites. Furthermore, when the genes were induced, GGR was slower in the transcribed regions than in the upstream regions. Finally, simultaneous deletion of RAD16, RAD26, and RPB9 resulted in no detectable repair in all sites along the region analyzed. Our results suggest that (a). TCR may be initiated by a transcription activator, presumably through the loading of RNA polymerase II, rather than by transcription initiation or elongation per se; (b). TCR and nucleosome disruption-enhanced GGR are the major causes of rapid repair in regions around and upstream of transcription start sites; (c). transcription machinery may hinder access of NER factors to a DNA lesion in the absence of a transcription-repair coupling factor; and (d). other than GGR mediated by Rad16 and TCR mediated by Rad26 and Rpb9, no other nucleotide excision repair pathway exists in these RNA polymerase II-transcribed genes.
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PMID:Dissecting transcription-coupled and global genomic repair in the chromatin of yeast GAL1-10 genes. 1473 64

The Escherichia coli FNR protein is a global transcription regulator that activates gene expression via interactions with the RNA polymerase alpha subunit C-terminal domain. Using preparations of E. coli RNA polymerase holoenzyme, specifically labelled with a DNA cleavage reagent, we have determined the location and orientation of the C-terminal domain of the RNA polymerase alpha subunit in transcriptionally competent complexes at a class II FNR-dependent promoter. We conclude that one alpha subunit C-terminal domain binds immediately upstream of FNR, and that its position and orientation is the same as at similar promoters dependent on CRP, another E. coli transcription activator that is related to FNR. In complementary experiments, we show that the second alpha subunit C-terminal domain of RNA polymerase can be repositioned by upstream-bound CRP, but not by upstream-bound FNR.
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PMID:Location of the Escherichia coli RNA polymerase alpha subunit C-terminal domain at an FNR-dependent promoter: analysis using an artificial nuclease. 1475 8

We describe the use of a bacterial two-hybrid system for the study of protein-protein interactions in Escherichia coli. This system is based on transcription activation and involves the synthesis of two fusion proteins within the bacterial cell whose interaction stimulates transcription of a reporter gene. Specifically, one of the fusion proteins can function as a transcription activator when its interaction partner is fused to a subunit of the bacterial RNA polymerase. This bacterial two-hybrid system has been used to study a number of interacting proteins from both prokaryotes and eukaryotes, and can be used to find interacting proteins from complex protein libraries.
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PMID:A bacterial two-hybrid system based on transcription activation. 1506 62

Recently determined structures of the Escherichia coli catabolite activator protein (CAP) in complex with DNA, and in complex with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) and DNA, have yielded insights into how CAP binds DNA and activates transcription. Comparison of multiple structures of CAP-DNA complexes has revealed the contributions of direct and indirect readout to DNA binding by CAP. The structure of the CAP-alphaCTD-DNA complex has provided the first structural description of interactions between a transcription activator and its functional target within the general transcription machinery. Using the structure of the CAP-alphaCTD-DNA complex, the structure of an RNA polymerase-DNA complex, and restraints from biophysical, biochemical and genetic experiments, it has been possible to construct detailed three-dimensional models of intact class I and class II transcription activation complexes.
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PMID:Catabolite activator protein: DNA binding and transcription activation. 1510 44

Wild-type transcriptional activation by Gcn4p is dependent on multiple coactivators, including SAGA, SWI/SNF, Srb mediator, CCR4-NOT, and RSC, which are all recruited by Gcn4p to its target promoters in vivo. It was not known whether these coactivators are required for assembly of the preinitiation complex (PIC) or for subsequent steps in the initiation or elongation phase of transcription. We find that mutations in subunits of these coactivators reduce the recruitment of TATA binding protein (TBP) and RNA polymerase II (Pol II) by Gcn4p at ARG1, ARG4, and SNZ1, implicating all five coactivators in PIC assembly at Gcn4p target genes. Recruitment of Pol II at SNZ1 and ARG1 was eliminated by mutations in TBP or by deletion of the TATA box, indicating that TBP binding is a prerequisite for Pol II recruitment by Gcn4p. However, several mutations in SAGA subunits and deletion of SRB10 had a greater impact on promoter occupancy of Pol II versus TBP, suggesting that SAGA and Srb mediator can promote Pol II binding independently of their stimulatory effects on TBP recruitment. Our results reveal an unexpected complexity in the cofactor requirements for the enhancement of PIC assembly by a single activator protein.
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PMID:An array of coactivators is required for optimal recruitment of TATA binding protein and RNA polymerase II by promoter-bound Gcn4p. 1512 33


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