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)

Transcriptional activation by the E.coli NtrC protein can occur via DNA looping between a DNA-bound activator and the target sigma(54) RNA polymerase. NtrC forms an octamer on DNA that is capable of binding two DNA molecules. Its ATPase activity is required for open complex formation. Geometric requirements for activation were assessed using a library of DNA bending sequences created by random ligation of A-tract oligonucleotides, as well as several designed sequences. Thirty random or designed sequences with a variety of DNA lengths and bending geometries were cloned in plasmids, and the library was used to replace the spacer between the NtrC binding sites and the core glnAp2 promoter. The activity of each promoter construct under nitrogen limitation was determined in vivo, in a lambda phage lacZ reporter system integrated as a single-copy lysogen to avoid titrating NtrC or polymerase. A wide variety of bending geometries was found to support a similar level of transcriptional activation ( approximately 3-4-fold). Computer modeling of the DNA trajectories suggests that the most inactive promoters have short spacer DNA and the NtrC sites on the opposite side of the helix as the wild-type sites; otherwise, the loop can form effectively. Flexibility and multivalency of the NtrC-Esigma(54) interaction apparently provides substantial independence from DNA stiffness constraints, and in general activation requires less efficient looping than repression. However, none of the random templates were as active as wild-type promoter. Subsidiary activator binding sites in the wild-type were found to be required for full activity, but, surprisingly, these sites could not be functionally replaced by strong binding sites. This suggests that one or more protomers in the NtrC octamer must form and then release contacts with DNA in order to complete the ATPase cycle and act as an AAA(+) activator of the Esigma(54). This dynamic DNA wrapping around the NtrC octamer is proposed to be necessary for efficient activation, and the wrapping may also reduce adventitious activation of other promoters.
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PMID:Geometric and dynamic requirements for DNA looping, wrapping and unwrapping in the activation of E.coli glnAp2 transcription by NtrC. 1532 47

SoxS is the transcription activator of the SoxRS regulon. Despite being synthesized de novo in response to oxidative stress and despite the large disparity between the number of SoxS binding sites and the number of SoxS molecules per cell, SoxS-dependent promoters are rapidly activated after the onset of the stress. With the usual recruitment/post-recruitment mechanisms being unsuitable for activating gene expression under these conditions, we previously proposed that SoxS functions by "pre-recruitment". In pre-recruitment, SoxS forms SoxS-RNA polymerase binary complexes, which use the DNA binding properties of SoxS and sigma(70) to distinguish SoxS-dependent promoters from housekeeping promoters and from the large number of sequence-equivalent but functionally irrelevant SoxS binding sites. With previous work in Escherichia coli having indicated that the most likely target on RNA polymerase for interaction with SoxS is the C-terminal domain of alpha, we investigated the interaction directly with the yeast two-hybrid system. We found that SoxS interacts with the alphaCTD and that SoxS positive control mutations disrupt the interaction. Moreover, single alanine substitutions of the alphaCTD that reduce or enhance SoxS activation in E.coli reduce or enhance the interaction between SoxS and the alphaCTD in yeast. Significantly, the critical amino acid residues lie in and around the DNA binding determinant of the alphaCTD, the first example of an activator contacting this determinant. These interactions were confirmed with an affinity immobilization assay. Lastly, we found that SoxS induction interferes with utilization of the UP element of an rRNA promoter. Thus, by functioning as a co-sigma factor that interacts with the DNA binding determinant of the alphaCTD, SoxS diverts RNA polymerase from UP-containing promoters to SoxS-activatable promoters.
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PMID:Novel protein--protein interaction between Escherichia coli SoxS and the DNA binding determinant of the RNA polymerase alpha subunit: SoxS functions as a co-sigma factor and redeploys RNA polymerase from UP-element-containing promoters to SoxS-dependent promoters during oxidative stress. 1546 42

In Escherichia coli, 6S RNA functions as a modulator of RNA polymerase sigma70-holoenzyme activity, but its biosynthetic pathway remains uncharacterized. In this study, to further understand the regulatory circuit of 6S RNA biosynthesis for the modulation of Esigma70 activity, we have characterized the biogenesis of 6S RNA. We reveal that there are two different precursors, a long and a short molecule, which are transcribed from the distal P2 and proximal P1 promoter, respectively. Transcription from the P2 promoter is both sigma70- and sigmaS-dependent, whereas, in contrast, P1 transcription is sigma70- but not sigmaS-dependent. Both precursors are processed to generate the 5' end of 6S RNA, and while the long precursor is processed exclusively by RNase E, the short precursor is processed by both RNase G and RNase E. Our data indicate that the switching of the utilization of both sigma factors and endoribonucleases in the biogenesis of 6S RNA would play an essential role in modulating its levels in E.coli.
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PMID:Regulation of 6S RNA biogenesis by switching utilization of both sigma factors and endoribonucleases. 1555 May 66

Anti-sigma70 factors interact with sigma70 proteins, the specificity subunits of prokaryotic RNA polymerase. The bacteriophage T4 anti-sigma70 protein, AsiA, binds tightly to regions 4.1 and 4.2 of the sigma70 subunit of Escherichia coli RNA polymerase and inhibits transcription from sigma70 promoters that require recognition of the canonical sigma70 -35 DNA sequence. In the presence of the T4 transcription activator MotA, AsiA also functions as a co-activator of transcription from T4 middle promoters, which retain the canonical sigma70 -10 consensus sequence but have a MotA box sequence centered at -30 rather than the sigma70 -35 sequence. The E.coli anti-sigma70 protein Rsd also interacts with region 4.2 of sigma70 and inhibits transcription from sigma70 promoters. Our sequence comparisons of T4 AsiA with Rsd, with the predicted AsiA orthologs of the T4-type phages RB69, 44RR, KVP40, and Aeh1, and with AlgQ, a regulator of alginate production in Pseudomonas aeruginosa indicate that these proteins share conserved amino acid residues at positions known to be important for the binding of T4 AsiA to sigma70 region 4. We show that, like T4 AsiA, Rsd binds to sigma70 in a native protein gel and, as with T4 AsiA, a L18S substitution in Rsd disrupts this complex. Previous work has assigned sigma70 amino acid F563, within region 4.1, as a critical determinant for AsiA binding. This residue is also involved in the binding of sigma70 to the beta-flap of core, suggesting that AsiA inhibits transcription by disrupting the interaction between sigma70 region 4.1 and the beta-flap. We find that as with T4 AsiA, the interaction of KVP40 AsiA, Rsd, or AlgQ with sigma70 region 4 is diminished by the substitution F563Y. We also demonstrate that like T4 AsiA and Rsd, KVP40 AsiA inhibits transcription from sigma70-dependent promoters. We speculate that the phage AsiA orthologs, Rsd, and AlgQ are members of a related family in T4-type phage and bacteria, which interact similarly with primary sigma factors. In addition, we show that even though a clear MotA ortholog has not been identified in the KVP40 genome and the phage genome appears to lack typical middle promoter sequences, KVP40 AsiA activates transcription from T4 middle promoters in the presence of T4 MotA. We speculate that KVP40 encodes a protein that is dissimilar in sequence, but functionally equivalent, to T4 MotA.
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PMID:A family of anti-sigma70 proteins in T4-type phages and bacteria that are similar to AsiA, a Transcription inhibitor and co-activator of bacteriophage T4. 1556 Nov 38

RNA chain elongation is a highly processive and accurate process that is finely regulated by numerous intrinsic and extrinsic signals. Here we describe a general mechanism that governs RNA polymerase (RNAP) movement and response to regulatory inputs such as pauses, terminators, and elongation factors. We show that E.coli RNAP moves by a complex Brownian ratchet mechanism, which acts prior to phosphodiester bond formation. The incoming substrate and the flexible F bridge domain of the catalytic center serve as two separate ratchet devices that function in concert to drive forward translocation. The adjacent G loop domain controls F bridge motion, thus keeping the proper balance between productive and inactive states of the elongation complex. This balance is critical for cell viability since it determines the rate, processivity, and fidelity of transcription.
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PMID:A ratchet mechanism of transcription elongation and its control. 1568 Mar 19

We have developed a method for transferring exogenous DNA molecules into isolated mammalian mitochondria using bacterial conjugation. In general, we accomplish this by (i) inserting an origin of DNA transfer (oriT) sequence into a DNA construct, (ii) transforming the construct into an appropriate Escherichia coli strain and then (iii) introducing the mobilizable DNA into mitochondria through conjugation. We tested this approach by transferring plasmid DNA containing a T7 promoter sequence into mitochondria that we had engineered to contain T7 RNA polymerase. After conjugation between E.coli and mitochondria, we detected robust levels of T7 transcription from the DNA constructs that had been transferred into the mitochondria. This approach for engineering DNA constructs in vitro and subsequent transfer into mitochondria by conjugation offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a potential route for transforming mitochondrial networks within mammalian cells.
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PMID:Transformation of isolated mammalian mitochondria by bacterial conjugation. 1615 61

The Bacillus cereus genome possesses three type IA topoisomerase genes. These genes, encoding DNA topoisomerase I and IIIalpha (bcTopo I, bcTopo IIIalpha), have been cloned into T7 RNA polymerase-regulated plasmid expression vectors and the enzymes have been overexpressed, purified and characterized. The proteins exhibit similar biochemical activity to their Escherichia coli counterparts, DNA topoisomerase I and III (ecTopo I, ecTopo III). bcTopo I is capable of efficiently relaxing negatively supercoiled DNA in the presence of Mg2+ but does not possess an efficient DNA decatenation activity. bcTopo IIIalpha is an active topoisomerase that is capable of relaxing supercoiled DNA at a broad range of Mg2+ concentrations; however, its DNA relaxation activity is not as efficient as that of bcTopo I. In addition, bcTopo III is a potent DNA decatenase that resolves oriC-based plasmid replication intermediates in vitro. Interestingly, bcTopo I and bcTopo IIIalpha are both able to compensate for the loss of ecTopo III in E.coli cells that lack ecTopo I. In contrast, ecTopo I cannot substitute for ecTopo III under these conditions.
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PMID:Bacillus cereus DNA topoisomerase I and IIIalpha: purification, characterization and complementation of Escherichia coli TopoIII activity. 1619 70

RNA polymerase from the mesophile Escherichia coli exists in two forms, the core enzyme and the holoenzyme. Using cryo-electron microscopy and single-particle analysis, we have obtained the structure of the complete RNA polymerase from E.coli containing the sigma54 factor within the closed-promoter complex. Comparisons with earlier reconstructions of the core enzyme and the sigma54 holoenzyme reveal the behaviour of this major variant RNA polymerase in defined functional states. The binding of DNA leads to significant conformational changes in the enzyme's catalytic subunits, apparently a necessity for the initiation of enhancer-dependent promoter-specific transcription.
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PMID:Conformational changes of Escherichia coli sigma54-RNA-polymerase upon closed-promoter complex formation. 1624 67

This work focuses on the experimental analysis of the time-course of protein expression in a cell-free system, in conjunction with the development of a computational model, denoted as progressive chain buildup (PCB), able to simulate translation kinetics and product formation as a function of starting reactant concentrations. Translation of the gene encoding the apomyoglobin (apoMb) model protein was monitored in an Escherichia coli cell-free system under different experimental conditions. Experimentally observed protein expression yields, product accumulation time-course and expression completion times match with the predictions by the PCB model. This algorithm regards elementary single-residue elongations as apparent second-order events and it accounts for aminoacyl-tRNA regeneration during translation. We have used this computational approach to model full-length protein expression and to explore the kinetic behavior of incomplete chains generated during protein biosynthesis. Most of the observed incomplete chains are non-obligatory dead-end species, in that their formation is not mandatory for full-length protein expression, and that they are unable to convert to the expected final translation product. These truncated polypeptides do not arise from post-translational degradation of full-length protein, but from a distinct subpopulation of chains which expresses intrinsically more slowly than the population leading to full-length product. The PCB model is a valuable tool to predict full-length and incomplete chain populations and formulate experimentally testable hypotheses on their origin. PCB simulations are applicable to E.coli cell-free expression systems (both in batch and dialysis mode) under the control of T7 RNA polymerase and to other environments where transcription and translation can be regarded as kinetically decoupled.
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PMID:Experimental and computational analysis of translation products in apomyoglobin expression. 1648 2

The Escherichia coli chromosome is condensed into an ill-defined structure known as the nucleoid. Nucleoid-associated DNA-binding proteins are involved in maintaining this structure and in mediating chromosome compaction. We have exploited chromatin immunoprecipitation and high-density microarrays to study the binding of three such proteins, FIS, H-NS and IHF, across the E.coli genome in vivo. Our results show that the distribution of these proteins is biased to intergenic parts of the genome, and that the binding profiles overlap. Hence some targets are associated with combinations of bound FIS, H-NS and IHF. In addition, many regions associated with FIS and H-NS are also associated with RNA polymerase.
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PMID:Association of nucleoid proteins with coding and non-coding segments of the Escherichia coli genome. 1696 79


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