Sigma70 Subunit Of RNA Polymerase Research Articles

Determinants of mer Promoter Activity from Pseudomonas aeruginosa.

Since the MerR family is known for its special regulatory mechanism, we aimed to explore which factors determine the expression activity of the mer promoter. The Tn501/Tn21 mer promoter contains an abnormally long spacer (19 bp) between the -35 and -10 elements, which is essential for the unique DNA distortion mechanism. To further understand the role of base sequences in the mer promoter spacer, this study systematically engineered a series of mutant derivatives and used luminescent and fluorescent reporter genes to investigate the expression activity of these derivatives. The results reveal that the expression activity of the mer promoter is synergistically modulated by the spacer length (17 bp is optimal) and the region upstream of -10 (especially -13G). The spacing is regulated by MerR transcription factors through symmetrical sequences, and -13G presumably functions through interaction with the RNA polymerase sigma-70 subunit.

Sequence-specific 1H, 15N, and 13C resonance assignments and relaxation parameters for the whole region 4 of Escherichia coli RNA polymerase sigma70 subunit in 10% TFE

Sequence-specific 1H, 15N, and 13C resonance assignments and relaxation parameters for the whole region 4 of Escherichia coli RNA polymerase sigma70 subunit in 10% TFE

Sequence-specific 1H, 15N, and 13C resonance assignments for the whole region 4 of Escherichia coli RNA polymerase sigma70 subunit

Sequence-specific 1H, 15N, and 13C resonance assignments for the whole region 4 of Escherichia coli RNA polymerase sigma70 subunit

Sequence-specific 1H, 15N, and 13C resonance assignments and relaxation parameters for the whole region 4 of Escherichia coli RNA polymerase sigma70 subunit in 30% TFE

Sequence-specific 1H, 15N, and 13C resonance assignments and relaxation parameters for the whole region 4 of Escherichia coli RNA polymerase sigma70 subunit in 30% TFE

Sequence-specific 1H, 15N, resonance assignments of nine 1H-{15N} HSQC spectras for the whole region 4 of Escherichia coli RNA polymerase sigma70 subunit recorded at TFE concentrations increasing in the range of 0-30%

Sequence-specific 1H, 15N, resonance assignments of nine 1H-{15N} HSQC spectras for the whole region 4 of Escherichia coli RNA polymerase sigma70 subunit recorded at TFE concentrations increasing in the range of 0-30%

Triad pattern algorithm for predicting strong promoter candidates in bacterial genomes.

BackgroundBacterial promoters, which increase the efficiency of gene expression, differ from other promoters by several characteristics. This difference, not yet widely exploited in bioinformatics, looks promising for the development of relevant computational tools to search for strong promoters in bacterial genomes.ResultsWe describe a new triad pattern algorithm that predicts strong promoter candidates in annotated bacterial genomes by matching specific patterns for the group I σ70 factors of Escherichia coli RNA polymerase. It detects promoter-specific motifs by consecutively matching three patterns, consisting of an UP-element, required for interaction with the α subunit, and then optimally-separated patterns of -35 and -10 boxes, required for interaction with the σ70 subunit of RNA polymerase. Analysis of 43 bacterial genomes revealed that the frequency of candidate sequences depends on the A+T content of the DNA under examination. The accuracy of in silico prediction was experimentally validated for the genome of a hyperthermophilic bacterium, Thermotoga maritima, by applying a cell-free expression assay using the predicted strong promoters. In this organism, the strong promoters govern genes for translation, energy metabolism, transport, cell movement, and other as-yet unidentified functions.ConclusionThe triad pattern algorithm developed for predicting strong bacterial promoters is well suited for analyzing bacterial genomes with an A+T content of less than 62%. This computational tool opens new prospects for investigating global gene expression, and individual strong promoters in bacteria of medical and/or economic significance.

High-resolution Crystal Structure of the Restriction–modification Controller Protein C.AhdI from Aeromonas hydrophila

Restriction-modification (R-M) systems serve to protect the host bacterium from invading bacteriophage. The multi-component system includes a methyltransferase, which recognizes and methylates a specific DNA sequence, and an endonuclease which recognises the same sequence and cleaves within or close to this site. The endonuclease will only cleave DNA that is unmethylated at the specific site, thus host DNA is protected while non-host DNA is cleaved. However, following DNA replication, expression of the endonuclease must be delayed until the host DNA is appropriately methylated. In many R-M systems, this regulation is achieved at the transcriptional level via the controller protein, or C-protein. We have solved the first X-ray structure of an R-M controller protein, C.AhdI, to 1.69 A resolution using selenomethionine MAD. C.AhdI is part of a Type IIH R-M system from the pathogen Aeromonas hydrophila. The structure reveals an all-alpha protein that contains a classical helix-turn-helix (HTH) domain and can be assigned to the Xre family of transcriptional regulators. Unlike its monomeric structural homologues, an extended helix generates an interface that results in dimerisation of the free protein. The dimer is electrostatically polarised and a positively charged surface corresponds to the position of the DNA recognition helices of the HTH domain. Comparison with the structure of the lambda cI ternary complex suggests that C.AhdI activates transcription through direct contact with the sigma70 subunit of RNA polymerase.

Amino acid contacts between sigma 70 domain 4 and the transcription activators RhaS and RhaR.

The RhaS and RhaR proteins are transcription activators that respond to the availability of L-rhamnose and activate transcription of the operons in the Escherichia coli L-rhamnose catabolic regulon. RhaR activates transcription of rhaSR, and RhaS activates transcription of the operon that encodes the L-rhamnose catabolic enzymes, rhaBAD, as well as the operon that encodes the L-rhamnose transport protein, rhaT. RhaS is 30% identical to RhaR at the amino acid level, and both are members of the AraC/XylS family of transcription activators. The RhaS and RhaR binding sites overlap the -35 hexamers of the promoters they regulate, suggesting they may contact the sigma70 subunit of RNA polymerase as part of their mechanisms of transcription activation. In support of this hypothesis, our lab previously identified an interaction between RhaS residue D241 and sigma70 residue R599. In the present study, we first identified two positively charged amino acids in sigma70, K593 and R599, and three negatively charged amino acids in RhaR, D276, E284, and D285, that were important for RhaR-mediated transcription activation of the rhaSR operon. Using a genetic loss-of-contact approach we have obtained evidence for a specific contact between RhaR D276 and sigma70 R599. Finally, previous results from our lab separately showed that RhaS D250A and sigma70 K593A were defective at the rhaBAD promoter. Our genetic loss-of-contact analysis of these residues indicates that they identify a second site of contact between RhaS and sigma70.

Efficient Inhibition of Escherichia Coli RNA Polymerase by the Bacteriophage T4 AsiA Protein Requires that AsiA Binds First to Free σ70

The bacteriophage T4 AsiA protein inhibits transcription from host and phage early promoters and is required, along with the T4 MotA protein, for activation of phage middle promoters. During infection, AsiA is found in a tight association with the sigma70 subunit of RNA polymerase. We show that AsiA binds rapidly to free sigma70 at either 4 degrees C or 30 degrees C to form an AsiA-sigma70 complex that with core efficiently reconstitutes the AsiA-inhibited RNA polymerase. In contrast, AsiA does not inhibit transcription after a 15 minute incubation with RNA polymerase holoenzyme at 4 degrees C, and at 30 degrees C an incubation of several minutes is required to inhibit most of the polymerase. We show that the heat step needed for AsiA is not the formation of an active AsiA protein. However, it is consistent with the momentary dissociation of holoenzyme to give free sigma70 and core. Our results indicate that AsiA is either unable to access holoenzyme directly or does so very slowly. Efficient generation of the AsiA-inhibited RNA polymerase requires that AsiA first binds to free sigma70 and then the AsiA-sigma70 complex binds to core to form the Asi-A-inhibited polymerase.

Conformational Changes of Escherichia coli RNA Polymerase ς70 Factor Induced by Binding to the Core Enzyme

Mutants of RNA polymerase sigma70 subunit from Escherichia coli with unique cysteine residues engineered into conserved region 1 (autoinhibition domain of sigma70), region 2.4 (-10 DNA element binding domain), region 4.2 (-35 DNA element binding domain), and a nonconserved region between regions 1 and 2 were prepared. The chemical reactivity of the cysteine at each position was determined for free sigma70 and sigma70 in complex with the core polymerase and was used as a measure of a conformational response of a particular region of the protein to an interaction with the core polymerase. Both increases and decreases in cysteine reactivity were observed in the presence of core polymerase at several positions in sigma70, providing direct physical evidence for modulation of sigma70 conformation by the core enzyme. Binding of the core polymerase resulted in increased solvent exposure of DNA binding domains of sigma70 and in more complex changes in the autoinhibition domain (region 1). Similar conformational changes in sigma70 were detected using fluorescence probes covalently attached to cysteine residues engineered into sigma70. Thus, the results obtained provided physical evidence supporting a model in which core enzyme allosterically regulates DNA binding activity of sigma70 by "unmasking" its DNA binding domains.

Ada Protein-RNA Polymerase ς Subunit Interaction and α Subunit-Promoter DNA Interaction Are Necessary at Different Steps in Transcription Initiation at the Escherichia coli adaandaidBPromoters

The methylated form of the Ada protein (meAda) binds the ada and aidB promoters between 60 and 40 base pairs upstream from the transcription start and activates transcription of the Escherichia coli ada and aidB genes. This region is also a binding site for the alpha subunit of RNA polymerase and resembles the rrnB P1 UP element in A/T content and location relative to the core promoter. In this report, we show that deletion of the C-terminal domain of the alpha subunit severely decreases meAda-independent binding of RNA polymerase to ada and aidB, affecting transcription initiation at these promoters. We provide evidence that meAda activates transcription by direct interaction with the C-terminal domain of RNA polymerase sigma70 subunit (amino acids 574-613). Several negatively charged residues in the sigma70 C-terminal domain are important for transcription activation by meAda; in particular, a glutamic acid to valine substitution at position 575 has a dramatic effect on meAda-dependent transcription. Based on these observations, we propose that the role of the alpha subunit at ada and aidB is to allow initial binding of RNA polymerase to the promoters. However, transcription initiation is dependent on meAda-sigma70 interaction.

Region 2.5 of the Escherichia coli RNA polymerase sigma70 subunit is responsible for the recognition of the 'extended-10' motif at promoters.

At some bacterial promoters, a 5'-TG-3' sequence element, located one base upstream of the -10 hexamer element, provides an essential motif necessary for transcription initiation. We have identified a mutant of the Escherichia coli RNA polymerase sigma70 subunit that has an altered preference for base sequences in this 'extended -10' region. We show that this mutant sigma70 subunit substantially increases transcription from promoters bearing 5'-TC-3' or 5'-TT-3' instead of a 5'-TG-3' motif, located one base upstream of the -10 hexamer. The mutant results from a single base pair substitution in the rpoD gene that causes a Glu to Gly change at position 458 of sigma70. This substitution identifies a functional region in sigma70 that is immediately adjacent to the well-characterized region 2.4 (positions 434-453, previously shown to contact the -10 hexamer). From these results, we conclude that this region (which we name region 2.5) is involved in contacting the 5'-TG-3' motif found at some bacterial promoters: thus, extended -10 regions are recognized by an extended region 2 of the RNA polymerase sigma70 subunit.

Changing the mechanism of transcriptional activation by phage lambda repressor.

The first steps of transcription initiation include binding of RNA polymerase to a promoter to form an inactive, unstable, closed complex (described by an equilibrium constant, K(B)) and isomerization of the closed complex to an active, stable, open complex (described by a forward rate constant, k(f)). lambda cI protein activates the PRM promoter by specifically increasing k(f). A positive control mutant, cI-pc2, is defective for activation because it fails to raise k(f). An Arg to His change in the sigma70 subunit of RNA polymerase was previously obtained as an allele-specific suppressor of cI-pc2. To elucidate how the mutant polymerase restores the activation function of the mutant activator, abortive initiation assays were performed, using purified cI proteins and RNA polymerase holoenzymes. The change in sigma does not significantly alter K(B) or k(f) in the absence of cI protein. As expected, cI-pc2 activates the mutant polymerase in the same way that wild-type cI activates the wild-type polymerase, by increasing k(f). An unexpected and novel finding is that the wild-type activator stimulates the mutant polymerase, but not wild-type polymerase, by increasing K(B).

An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription.

The bacteriophage T4 MotA protein is a transcriptional activator of T4-modified host RNA polymerase and is required for activation of the middle class of T4 promoters. MotA alone binds to the -30 region of T4 middle promoters, a region that contains the MotA box consensus sequence [(t/a)(t/a)TGCTT(t/c)A]. We report the isolation and characterization of a protein designated Mot21, in which the first 8 codons of the wild-type motA sequence have been replaced with 11 different codons. In gel retardation assays, Mot21 and MotA bind DNA containing the T4 middle promoter P(uvsX) similarly, and the proteins yield similar footprints on P(uvsX). However, Mot21 is severely defective in the activation of transcription. On native protein gels, a new protein species is seen after incubation of the sigma70 subunit of RNA polymerase and wild-type MotA protein, suggesting a direct protein-protein contact between MotA and sigma70. Mot21 fails to form this complex, suggesting that this interaction is necessary for transcriptional activation and that the Mot21 defect arises because Mot21 cannot form this contact like the wild-type activator.