RNA Polymerase Binding Site Research Articles

Catabolism of Phenylalanine by Pseudomonas putida: The NtrC-family PhhR Regulator Binds to Two Sites Upstream from the phhA Gene and Stimulates Transcription with σ 70

Pseudomonas putida uses l-phenylalanine as the sole nitrogen source for growth by converting l-phenylalanine to l-tyrosine, which acts as a donor of the amino group. This metabolic step requires the products of the phhA and phhB genes, which form an operon. Expression of the phhA promoter is mediated by the phhR gene product in the presence of l-phenylalanine or l-tyrosine. The PhhR protein belongs to the NtrC family of enhancers. In contrast with most members of this family of regulators, transcription from the promoter of the phhAB operon (P phhA ) is mediated by RNA polymerase with σ 70 rather than with σ 54. The PhhR regulator binds two similar but non-identical upstream PhhR motifs (5′-TGTAAAATTATCGTTACG-3′ and 5′-ACAAAAACTGTGTTTCCG-3′) that are located 39 and 97 nucleotides upstream of the proposed −35 hexamer for RNA polymerase, respectively. These motifs are called PhhR proximal and PhhR distal binding motifs because of their position with respect to the RNA polymerase binding site. Affinity of PhhR for its target sequences was determined by isothermal titration calorimetry and was found to be around 30 nM for the proximal site and 2 μM for the distal site, and the binding stoichiometry is of a dimer per binding site. Both target sequences are sine qua non requirements for transcription, since inactivation of either of them resulted in no transcription from the phhA promoter. An IHF binding site overlaps the proximal PhhR proximal motif, which is recognized by IHF with a K D of around 1.2 μM. IHF may consequently compete with PhhR for binding and indeed inhibits PhhR-dependent phhAB operon expression.

Transcription linked to recombination: a gene-internal promoter coincides with the recombination hot spot II of the human MLL gene

The MLL gene is frequently involved in chromosomal translocations associated with high-risk acute leukaemia. Infant and therapy-related acute leukaemia patients display chromosomal breakpoints preferentially clustered in the telomeric portion of the MLL breakpoint cluster region (SCII). Here, we demonstrate that SCII colocalizes with a gene-internal promoter element in the mouse and human MLL gene, respectively. The mRNA generated encodes an N-terminally truncated version of MLL that still exhibits many functional regions, including the C-terminal SET-domain. Etoposide-induced DNA double-strand breaks colocalize with the binding site of RNA polymerase II and the transcription initiation region, but not with a nearby Topo II consensus sequence. Thus, the observed genomic instability of the human MLL gene is presumably linked to transcriptional processes. The consequences of this novel finding for the creation of chromosomal translocations, the biology of the MLL protein and for MLL-mediated acute leukaemia are discussed.

Mutational analysis of intervening sequences connecting the binding sites for integration host factor, PepA, PurR, and RNA polymerase in the control region of the Escherichia coli carAB operon, encoding carbamoylphosphate synthase.

Transcription of the carAB operon encoding the unique carbamoylphosphate synthase of Escherichia coli reflects the dual function of carbamoylphosphate in the biosynthesis of arginine and pyrimidine nucleotides. The tandem pair of promoters is regulated by various mechanisms depending on the needs of both pathways and the maintenance of a pyrimidine/purine nucleotide balance. Here we focus on the linker regions that impose the distribution of target sites for DNA-binding proteins involved in pyrimidine- and purine-specific repression of the upstream promoter P1. We introduced deletions and insertions, and combinations thereof, in four linkers connecting the binding sites for integration host factor (IHF), PepA, PurR, and RNA polymerase and studied the importance of phasing and spacing of the targets and the importance of the nucleotide sequence of the linkers. The two PepA binding sites must be properly aligned and separated with respect to each other and to the promoter for both pyrimidine- and purine-mediated repression. Similarly, the phasing and spacing of the IHF and PEPA2 sites are strictly constrained but only for pyrimidine-specific repression. The IHF target is even dispensable for purine-mediated regulation. Thus, a correct localization of PepA within the higher-order nucleoprotein complex is a prerequisite for the establishment of pyrimidine-mediated repression and for the coupling between purine- and pyrimidine-dependent regulation. Our data also suggest the existence of a novel cis-acting pyrimidine-specific regulatory target located around position -60. Finally, the analysis of a P1 derivative devoid of its control region has led to a reappraisal of the effect of excess adenine on P1 and has revealed that P1 has no need for a UP element.

MarA-mediated Transcriptional Repression of the rob Promoter

The Escherichia coli transcriptional regulator MarA affects functions that include antibiotic resistance, persistence, and survival. MarA functions as an activator or repressor of transcription utilizing similar degenerate DNA sequences (marboxes) with three different binding site configurations with respect to the RNA polymerase-binding sites. We demonstrate that MarA down-regulates rob transcripts both in vivo and in vitro via a MarA-binding site within the rob promoter that is positioned between the -10 and -35 hexamers. As for the hdeA and purA promoters, which are repressed by MarA, the rob marbox is also in the "backward" orientation. Protein-DNA interactions show that SoxS and Rob, like MarA, bind the same marbox in the rob promoter. Electrophoretic mobility shift analyses with a MarA-specific antibody demonstrate that MarA and RNA polymerase form a ternary complex with the rob promoter DNA. Transcription experiments in vitro and potassium permanganate footprinting analysis show that MarA affects the RNA polymerase-mediated closed to open complex formation at the rob promoter.

Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors

Members of the IclR family of regulators are proteins with around 250 residues. The IclR family is best defined by a profile covering the effector binding domain. This is supported by structural data and by a number of mutants showing that effector specificity lies within a pocket in the C-terminal domain. These regulators have a helix-turn-helix DNA binding motif in the N-terminal domain and bind target promoters as dimers or as a dimer of dimers. This family comprises regulators acting as repressors, activators and proteins with a dual role. Members of the IclR family control genes whose products are involved in the glyoxylate shunt in Enterobacteriaceae, multidrug resistance, degradation of aromatics, inactivation of quorum-sensing signals, determinants of plant pathogenicity and sporulation. No clear consensus exists on the architecture of DNA binding sites for IclR activators: the MhpR binding site is formed by a 15-bp palindrome, but the binding sites of PcaU and PobR are three perfect 10-bp sequence repetitions forming an inverted and a direct repeat. IclR-type positive regulators bind their promoter DNA in the absence of effector. The mechanism of repression differs among IclR-type regulators. In most of them the binding sites of RNA polymerase and the repressor overlap, so that the repressor occludes RNA polymerase binding. In other cases the repressor binding site is distal to the RNA polymerase, so that the repressor destabilizes the open complex.

Immobilization of Escherichia coli RNA polymerase and location of binding sites by use of chromatin immunoprecipitation and microarrays.

The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the beta' subunit. The DNA was amplified and hybridized to "tiled" oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known sigma70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique.

The Silencing of Pseudogenes

Pseudogenes are nonfunctional DNA sequences that can accumulate in the genomes of some bacterial species, especially those undergoing processes like niche change, host specialization, or weak selection strength. They may last for long evolutionary periods, opening the question of how the genome prevents expression of these degenerated or disrupted genes that would presumably give rise to malfunctioning proteins. We have investigated ribosomal binding strength at Shine-Dalgarno sequences and the prevalence of sigma70 promoter regions in pseudogenes across bacteria. It is reported that the RNA polymerase-binding sites and more strongly the ribosome-binding regions of pseudogenes are highly degraded, suggesting that transcription and translation are impaired in nonfunctional open reading frames. This would reduce the metabolic investment on faulty proteins because although pseudogenes can persist for long time periods, they would be effectively silenced. It is unclear whether mutation accumulation on regulatory regions is neutral or whether it is accelerated by selection.

Identification of amino acid residues of the Agrobacterium tumefaciens quorum‐sensing regulator TraR that are critical for positive control of transcription

The LuxR-type quorum-sensing transcription factor TraR regulates replication and conjugal transfer of the tumour-inducing (Ti) plasmid in the plant pathogen Agrobacterium tumefaciens. TraR is a two-domain protein with an N-terminal domain that binds to the quorum-sensing signal N-3-oxooctanoyl- l-homoserine lactone (OOHL) and a C-terminal domain that binds to specific DNA sequences called tra boxes. TraR-OOHL complexes form homodimers that activate transcription of at least seven promoters on the Ti plasmid. At five promoters, a tra box overlaps the binding site of core RNA polymerase (class II promoters), while in the other two promoters, this site is located farther upstream (class I promoters). In this study, we performed saturating point mutagenesis of the surface residues of the TraR C-terminal domain. Each mutant was tested for proteolytic stability and transcription activity in vivo, and for DNA binding activity in vitro. Mutants of TraR with single substitutions at positions W184, V187, K189, E193Q, V197 and D217 have wild-type levels of accumulation and DNA binding, but are defective in transcription of both types of promoters. These residues constitute a patch on the surface of the DNA-binding domain. We propose that this patch is an activating region that recruits RNA polymerase to TraR-dependent promoters through direct contact. As residues of this patch are critical for activation at both a class I and a class II promoter, we predict that these residues may contact the C-terminal domain of the RNA polymerase alpha-subunit.

Supportive and inhibitory elements of a putative PrfA‐dependent promoter inListeria monocytogenes

Elements essential for PrfA-dependent transcription were analysed on two promoters of Listeria monocytogenes, the PrfA-dependent promoter of the phospholipase gene plcA (PplcA) and a putative promoter of the aroA gene (ParoA2) which contains a similar PrfA-binding site and a similar -10 box as PplcA but does not function as PrfA-dependent promoter. We constructed a series of hybrid plcA-aroA promoters by exchanging corresponding sequence elements of these two 'promoters'. The results showed that the two critical elements of PrfA-dependent promoters, the PrfA-box and the -10 box, can be functionally exchanged as long as the distance in between is maintained to 22 or 23 bp. However, the interspace sequence and the sequence downstream of the -10 box of ParoA2 were strongly inhibitory for PrfA-dependent transcription. A detailed analysis of these two sequences revealed that the RNA polymerase binding site being part of the actual in vivo and in vitro used aroA promoter (ParoA1) and a sequence immediately downstream of the putative -10 site, possibly blocking the formation of the open complex, were responsible for the inhibition of PrfA-dependent transcription from ParoA2. Taking into consideration the lessons learned from this study we were able to construct a functional PrfA-dependent aroA promoter.

A new PCR-based approach for the preparation of RNA probe

A number of PCR-based in situ hybridization (ISH) techniques have been reported to facilitate the procedure. However, those techniques require additional gene specific primers with RNA polymerase binding site. We developed a new PCR-based ISH technique without extra gene-specific primers. We amplified gene specific PCR products with regular gene-specific primer pairs. Special linker, including T7 RNA polymerase binding site, was adapted to amplified PCR products. Secondary PCR was performed with T7 primer, and forward or reverse primer, used for the first PCR to prepare template DNA for RNA transcription. We were able to generate sense and anti-sense probes for ISH in a day. Recently, real-time PCR and ISH are required to validate microarray results quantitatively and qualitatively. This technique can be expected to facilitate the high-throughput validation of transcripts detected by microarrays.

E4F1, a Novel Estrogen-Responsive Gene in Possible Atheroprotection, Revealed by Microarray Analysis

Estrogen has been postulated to be involved in inhibition of vascular smooth muscle cell (VSMC) proliferation mainly via estrogen receptor (ER), but the detailed mechanism has remained primarily unknown. Therefore, in this study, microarray analysis was used in two types of cultured human VSMCs: one positive for ER alpha, and the other for ER beta, which were treated by estrogens to detect the estrogen-responsive genes. We also used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate mRNA levels of selective target gene (TG) in these cells. We further studied whether the TG product was involved in inhibition of proliferation using small interfering RNA (siRNA) of the TG transfection. We subsequently used quantitative RT-PCR and in situ hybridization analysis to evaluate the expression of these gene products in human aorta. E4F1, a possible inducer of cell growth arrest, was markedly increased only in ER alpha-positive VSMCs by estrogens in both microarray and RT-PCR analyses. Blocking of E4F1 using siRNA suppressed estrogenic inhibition of ER alpha-positive VSMC proliferation. E4F1 mRNA was abundant in premenopausal female aorta with mild atherosclerotic changes. E4F1 is therefore considered one of the estrogen-responsive genes involving ER alpha-mediated inhibition of VSMC proliferation and may play an important role in estrogen-related atheroprotection of human aorta.

Predicting gene regulation by sigma factors in Bacillus subtilis from genome-wide data.

Sigma factors regulate the expression of genes in Bacillus subtilis at the transcriptional level. We assess the accuracy of a fold-change analysis, Bayesian networks, dynamic models and supervised learning based on coregulation in predicting gene regulation by sigma factors from gene expression data. To improve the prediction accuracy, we combine sequence information with expression data by adding their log-likelihood scores and by using a logistic regression model. We use the resulting score function to discover currently unknown gene regulations by sigma factors. The coregulation-based supervised learning method gave the most accurate prediction of sigma factors from expression data. We found that the logistic regression model effectively combines expression data with sequence information. In a genome-wide search, highly significant logistic regression scores were found for several genes whose transcriptional regulation is currently unknown. We provide the corresponding RNA polymerase binding sites to enable a straightforward experimental verification of these predictions.

Mode of action of the TyrR protein: repression and activation of the tyrP promoter of Escherichia coli.

The tyrP gene of Escherichia coli encodes a tyrosine specific transporter. Its synthesis is repressed by tyrosine but is activated by phenylalanine and to a lesser extent by tryptophan. Both of these effects are mediated by the TyrR protein when it binds to one or both of its cognate binding sites (TyrR boxes) which encompass nucleotides -30 to -75. Activation in the presence of phenylalanine or tryptophan involves a dimer binding to the upstream box and interacting with the alpha subunit (alphaCTD) of RNA polymerase (RNAP). Repression in the presence of tyrosine involves a hexamer binding to both TyrR boxes. The molecular basis for this repression has been studied in vitro. Whereas initial gel shift experiments fail to show the exclusion of RNAP from the promoter region when TyrR hexamer is bound, a DNase I analysis of slices from the gel shows that in the presence of TyrR, RNAP now binds to a previously unrecognized upstream promoter. Although this upstream promoter is bound strongly by RNAP and forms an open complex on linear DNA templates, it fails to form an open complex on supercoiled templates in vitro and is unable to initiate transcription in vivo. A subsequent gel shift assay using a tyrP fragment which eliminates the upstream RNAP binding site confirms conclusively that, in the presence of tyrosine and ATP, the TyrR protein prevents RNAP from binding to the tyrP promoter. In vitro studies have also been carried out in the presence of TyrR protein and phenylalanine. Binding of TyrR protein to the upstream TyrR box in the presence of phenylalanine is shown to increase the affinity of RNAP for the promoter and stimulate open complex formation at the -10 region of the tyrP promoter. This observation coupled with the results from mutational analysis supports the proposal that TyrR-phenylalanine activates tyrP transcription by stimulating the onset of open complex formation.

Sigma70 Promoters in Escherichia coli: Specific Transcription in Dense Regions of Overlapping Promoter-like Signals

We present here a computational analysis showing that sigma70 house-keeping promoters are located within zones with high densities of promoter-like signals in Escherichia coli, and we introduce strategies that allow for the correct computer prediction of sigma70 promoters. Based on 599 experimentally verified promoters of E. coli K-12, we generated and evaluated more than 200 weight matrices optimizing different criteria to obtain the best recognition matrices. The alignments generating the best statistical models did not fully correspond with the canonical sigma70 model. However, matrices that correspond to such a canonical model performed better as tools for prediction. We tested the predictive capacity of these matrices on 250 bp long regions upstream of gene starts, where 90% of the known promoters occur. The computational matrix models generated an average of 38 promoter-like signals within each 250 bp region. In more than 50% of the cases, the true promoter does not have the best score within the region. We observed, in fact, that real promoters occur mostly within regions with high densities of overlapping putative promoters. We evaluated several strategies to identify promoters. The best one uses an intrinsic score of the −10 and −35 hexamers that form the promoter as well as an extrinsic score that uses the distribution of promoters from the start of the gene. We were able to identify 86% true promoters correctly, generating an average of 4.7 putative promoters per region as output, of which 3.7, on average, exist in clusters, as a series of overlapping potentially competing RNA polymerase-binding sites. As far as we know, this is the highest predictive capability reported so far. This high signal density is found mainly within regions upstream of genes, contrasting with coding regions and regions located between convergently transcribed genes. These results are consistent with experimental evidence that show the existence of multiple overlapping promoter sites that become functional under particular conditions. This density is probably the consequence of a rich number of vestiges of promoters in evolution. We suggest that transcriptional regulators as well as other functional promoters play an important role in keeping these latent signals suppressed.

Global amplification of sense RNA: a novel method to replicate and archive mRNA for gene expression analysis

We have developed a procedure to amplify mRNA into sense RNA (sRNA) so as to create a regenerating biorepository representing the complex mRNA profile in the original sample. The procedure exploits the template-switching activity of reverse transcriptase to incorporate RNA polymerase binding sites upstream of single-stranded cDNA (ss cDNA). Limited PCR was used for double-stranded DNA (dsDNA) synthesis. sRNA was synthesized from PCR products by in vitro transcription (IVT). sRNA was evaluated by real-time reverse transcription (RT)-PCR. sRNA synthesis was successful with RNA from human cell lines and tissues, yielding 2000- to 2500-fold amplification of glyceraldeyde-3 phosphate dehydrogenase (G3PDH). The size of sRNA ranged from 3.0 to 0.1 kb. sRNA synthesis preserved the relative differences in plant mRNAs spiked at abundance ranging over 5 orders of magnitude (0.00001–0.1%). This reflects the high fidelity of sRNA synthesis for mRNA as low as 0.3 copies/cell. sRNA is amplified synthetic mRNA in the 5′→3′ direction; the appropriate template for any gene expression analysis.

The Bacillus subtilis transition state regulator AbrB binds to the -35 promoter region of comK.

Genetic competence is a differentiation process initiated by Bacillus subtilis as a result of nutritional deprivation, and is controlled by a complex signal transduction cascade. The promoter of comK, encoding the competence transcription factor, is regulated by at least four different transcription factors: Rok, CodY, DegU and ComK itself. Genetic data have shown that comK expression is influenced by the transition state regulator AbrB as well. In this paper we show that AbrB binds specifically to the comK promoter and covers the RNA polymerase binding site, making it the fifth transcription factor regulating the activity of the comK promoter.

Improving the predictive value of the competence transcription factor (ComK) binding site in Bacillus subtilis using a genomic approach

Generally, the presence of a consensus sequence in the promoter of a gene is taken as indication for regulation by the transcription factor that binds to this sequence. In light of the recent developments in genome research, we were interested to what extent this supposition is valid. We examined the relationship between the presence of a binding site for ComK, the competence transcription factor of Bacillus subtilis, and actual transcriptional activation by ComK. Bacillus subtilis contains 1062 putative ComK-binding sites (K-boxes) in its genome. We employed DNA macroarrays to identify ComK-activated genes, and found that the presence of a K-box is an unreliable predictor for regulation. Only approximately 8% of the genes containing a K-box in the putative promoter region are regulated by ComK. The predictive value of a K-box could be improved by taking into consideration the degree of deviation from the K-box consensus sequence, the presence of extra ComK-binding motifs and the positions of RNA polymerase-binding sites. Finally, many of the ComK-activated genes show no apparent function related to the competence process. Based on our findings, we propose that the ComK-dependent activation of several genes might serve no biological purpose and can be considered 'evolutionary noise'.

The Bacillus subtilis transition state regulator AbrB binds to the −35 promoter region of comK

Genetic competence is a differentiation process initiated by Bacillus subtilis as a result of nutritional deprivation, and is controlled by a complex signal transduction cascade. The promoter of comK, encoding the competence transcription factor, is regulated by at least four different transcription factors: Rok, CodY, DegU and ComK itself. Genetic data have shown that comK expression is influenced by the transition state regulator AbrB as well. In this paper we show that AbrB binds specifically to the comK promoter and covers the RNA polymerase binding site, making it the fifth transcription factor regulating the activity of the comK promoter.

FleQ, the major flagellar gene regulator in Pseudomonas aeruginosa, binds to enhancer sites located either upstream or atypically downstream of the RpoN binding site.

In Pseudomonas aeruginosa, flagellar genes are regulated in a cascade headed by FleQ, an NtrC/NifA-type activator. FleQ and RpoN positively regulate expression of flhA, fliE, fliL, and fleSR genes, among others. Direct interaction of FleQ with flhA, fliE, fliL, and fleSR promoters was demonstrated by gel shift assay, along with experiments to conclusively determine the specificity of its binding. DNase I footprinting was performed to determine the FleQ binding sites on flhA, fliE, fliL, and fleSR promoters. No sequence conservation among these binding sites was observed. Primer extension analysis revealed the transcription start sites (TSSs) to be localized above the FleQ binding sites in flhA, fliE, and fliL promoters. Analysis of the above data revealed FleQ binding to be in the leader sequence of these promoters, whereas FleQ binding was 67 bp upstream of the TSS in the fleSR promoter. Mutagenesis of the FleQ binding site in the flhA promoter confirmed its functionality in vivo. Deletion of the flhA promoter upstream of the RNA polymerase binding site did not result in a significant loss of promoter activity. These results point to two modes of regulation by an NtrC-type regulator in the flagellar hierarchy in P. aeruginosa, the first being the typical model of activation from a distance via looping in the fleSR promoter and the second involving flhA, fliE, and fliL promoters, where FleQ binds in the downstream vicinity of the promoter and activates transcription without looping.

Regulation of Salmonella enterica serovar Typhimurium mntH transcription by H(2)O(2), Fe(2+), and Mn(2+).

MntH, a bacterial homolog of mammalian natural resistance associated macrophage protein 1 (Nramp1), is a primary transporter for Mn(2+) influx in Salmonella enterica serovar Typhimurium and Escherichia coli. S. enterica serovar Typhimurium MntH contributes to H(2)O(2) resistance and is important for full virulence. Consistent with its phenotype and function, mntH is regulated at the transcriptional level by both H(2)O(2) and substrate cation. We have now identified three trans-acting regulatory factors and the three corresponding cis-acting mntH promoter motifs that mediate this regulation. In the presence of hydrogen peroxide, mntH is activated by OxyR, acting through an OxyR-binding motif centered just upstream of the likely -35 RNA polymerase-binding site. In the presence of Fe(2+), mntH is repressed primarily by Fur, acting through a Fur-binding motif overlapping the -35 region. In the presence of Mn(2+), mntH is repressed primarily by the Salmonella equivalent of E. coli b0817, a distant homolog of the Bacillus subtilis manganese transport repressor, MntR, acting through an inverted-repeat motif located between the likely -10 polymerase binding site and the ribosome binding site. E. coli b0817 was recently shown to bind the identical inverted-repeat motif in the E. coli mntH promoter and hence has been renamed MntR (S. I. Patzer and K. Hantke, J. Bacteriol. 183:4806-4813, 2001). Using Deltafur, DeltamntR, and Deltafur DeltamntR mutant strains as well as mutations in the Fur- and MntR-binding motif elements, we found that Fe(2+) can also mediate repression through the Mn(2+) repressor MntR.