Transcription Initiation In Bacteria Research Articles

Lisen&Curate: A platform to facilitate gathering textual evidence for curation of regulation of transcription initiation in bacteria

The number of published papers in biomedical research makes it rather impossible for a researcher to keep up to date. This is where manually curated databases contribute facilitating the access to knowledge. However, the structure required by databases strongly limits the type of valuable information that can be incorporated. Here, we present Lisen&Curate, a curation system that facilitates linking sentences or part of sentences (both considered sources) in articles with their corresponding curated objects, so that rich additional information of these objects is easily available to users. These sources are going to be offered both within RegulonDB and a new database, L-Regulon. To show the relevance of our work, two senior curators performed a curation of 31 articles on the regulation of transcription initiation of E. coli using Lisen&Curate. As a result, 194 objects were curated and 781 sources were recorded. We also found that these sources are useful to develop automatic approaches to detect objects in articles by observing word frequency patterns and by carrying out an open information extraction task. Sources may help to elaborate a controlled vocabulary of experimental methods. Finally, we discuss our ecosystem of interconnected applications, RegulonDB, L-Regulon, and Lisen&Curate, to facilitate the access to knowledge on regulation of transcription initiation in bacteria. We see our proposal as the starting point to change the way experimentalists connect a piece of knowledge with its evidence using RegulonDB.

Diverse and unified mechanisms of transcription initiation in bacteria.

Transcription of DNA is a fundamental process in all cellular organisms. The enzyme responsible for transcription, RNA polymerase, is conserved in general architecture and catalytic function across the three domains of life. Diverse mechanisms are used among and within the different branches to regulate transcription initiation. Mechanistic studies of transcription initiation in bacteria are especially amenable because the promoter recognition and melting steps are much less complicated than in eukaryotes or archaea. Also, bacteria have critical roles in human health as pathogens and commensals, and the bacterial RNA polymerase is a proven target for antibiotics. Recent biophysical studies of RNA polymerases and their inhibition, as well as transcription initiation and transcription factors, have detailed the mechanisms of transcription initiation in phylogenetically diverse bacteria, inspiring this Review to examine unifying and diverse themes in this process.

Where to begin? Sigma factors and the selectivity of transcription initiation in bacteria.

Transcription is the fundamental process that enables the expression of genetic information. DNA-directed RNA polymerase (RNAP) uses one strand of the DNA duplex as template to produce complementary RNA molecules that serve in translation (rRNA, tRNA), protein synthesis (mRNA) and regulation (sRNA). Although the RNAP core is catalytically competent for RNA synthesis, the selectivity of transcription initiation requires a sigma (σ) factor for promoter recognition and opening. Expression of alternative σ factors provides a powerful mechanism to control the expression of discrete sets of genes (a σ regulon) in response to specific nutritional, developmental or stress-related signals. Here, I review the key insights that led to the original discovery of σ factor 50years ago and the subsequent discovery of alternative σ factors as a ubiquitous mechanism of bacterial gene regulation. These studies form a prelude to the more recent, genomics-enabled insights into the vast diversity of σ factors in bacteria.

Investigation of specific interactions between T7 promoter and T7 RNA polymerase by force spectroscopy using atomic force microscope.

The specific recognition and binding of promoter and RNA polymerase is the first step of transcription initiation in bacteria and largely determines transcription activity. Therefore, direct analysis of the interaction between promoter and RNA polymerase in vitro may be a new strategy for promoter characterization, to avoid interference due to the cell's biophysical condition and other regulatory elements. In the present study, the specific interaction between T7 promoter and T7 RNA polymerase was studied as a model system using force spectroscopy based on atomic force microscope (AFM). The specific interaction between T7 promoter and T7 RNA polymerase was verified by control experiments, and the rupture force in this system was measured as 307.2 ± 6.7 pN. The binding between T7 promoter mutants with various promoter activities and T7 RNA polymerase was analyzed. Interaction information including rupture force, rupture distance and binding percentage were obtained in vitro, and reporter gene expression regulated by these promoters was also measured according to a traditional promoter activity characterization method in vivo Using correlation analysis, it was found that the promoter strength characterized by reporter gene expression was closely correlated with rupture force and the binding percentage by force spectroscopy. These results indicated that the analysis of the interaction between promoter and RNA polymerase using AFM-based force spectroscopy was an effective and valid approach for the quantitative characterization of promoters.

Physiological roles of sigma factor SigD in Corynebacterium glutamicum

BackgroundSigma factors are one of the components of RNA polymerase holoenzymes, and an essential factor of transcription initiation in bacteria. Corynebacterium glutamicum possesses seven genes coding for sigma factors, most of which have been studied to some detail; however, the role of SigD in transcriptional regulation in C. glutamicum has been mostly unknown.ResultsIn this work, pleiotropic effects of sigD overexpression at the level of phenotype, transcripts, proteins and metabolites were investigated. Overexpression of sigD decreased the growth rate of C. glutamicum cultures, and induced several physiological effects such as reduced culture foaming, turbid supernatant and cell aggregation. Upon overexpression of sigD, the level of Cmt1 (corynomycolyl transferase) in the supernatant was notably enhanced, and carbohydrate-containing compounds were excreted to the supernatant. The real-time PCR analysis revealed that sigD overexpression increased the expression of genes related to corynomycolic acid synthesis (fadD2, pks), genes encoding corynomycolyl transferases (cop1, cmt1, cmt2, cmt3), L, D-transpeptidase (lppS), a subunit of the major cell wall channel (porH), and the envelope lipid regulation factor (elrF). Furthermore, overexpression of sigD resulted in trehalose dicorynomycolate accumulation in the cell envelope.ConclusionsThis study demonstrated that SigD regulates the synthesis of corynomycolate and related compounds, and expanded the knowledge of regulatory functions of sigma factors in C. glutamicum.

Global repositioning of transcription start sites in a plant-fermenting bacterium.

Bacteria respond to their environment by regulating mRNA synthesis, often by altering the genomic sites at which RNA polymerase initiates transcription. Here, we investigate genome-wide changes in transcription start site (TSS) usage by Clostridium phytofermentans, a model bacterium for fermentation of lignocellulosic biomass. We quantify expression of nearly 10,000 TSS at single base resolution by Capp-Switch sequencing, which combines capture of synthetically capped 5′ mRNA fragments with template-switching reverse transcription. We find the locations and expression levels of TSS for hundreds of genes change during metabolism of different plant substrates. We show that TSS reveals riboswitches, non-coding RNA and novel transcription units. We identify sequence motifs associated with carbon source-specific TSS and use them for regulon discovery, implicating a LacI/GalR protein in control of pectin metabolism. We discuss how the high resolution and specificity of Capp-Switch enables study of condition-specific changes in transcription initiation in bacteria.

Local and global regulation of transcription initiation in bacteria.

Gene expression in bacteria relies on promoter recognition by the DNA-dependent RNA polymerase and subsequent transcription initiation. Bacterial cells are able to tune their transcriptional programmes to changing environments, through numerous mechanisms that regulate the activity of RNA polymerase, or change the set of promoters to which the RNA polymerase can bind. In this Review, we outline our current understanding of the different factors that direct the regulation of transcription initiation in bacteria, whether by interacting with promoters, with RNA polymerase or with both, and we discuss the diverse molecular mechanisms that are used by these factors to regulate gene expression.

Region 4 of Rhizobium etli Primary Sigma Factor (SigA) Confers Transcriptional Laxity in Escherichia coli.

Sigma factors are RNA polymerase subunits engaged in promoter recognition and DNA strand separation during transcription initiation in bacteria. Primary sigma factors are responsible for the expression of housekeeping genes and are essential for survival. RpoD, the primary sigma factor of Escherichia coli, a γ-proteobacteria, recognizes consensus promoter sequences highly similar to those of some α-proteobacteria species. Despite this resemblance, RpoD is unable to sustain transcription from most of the α-proteobacterial promoters tested so far. In contrast, we have found that SigA, the primary sigma factor of Rhizobium etli, an α-proteobacteria, is able to transcribe E. coli promoters, although it exhibits only 48% identity (98% coverage) to RpoD. We have called this the transcriptional laxity phenomenon. Here, we show that SigA partially complements the thermo-sensitive deficiency of RpoD285 from E. coli strain UQ285 and that the SigA region σ4 is responsible for this phenotype. Sixteen out of 74 residues (21.6%) within region σ4 are variable between RpoD and SigA. Mutating these residues significantly improves SigA ability to complement E. coli UQ285. Only six of these residues fall into positions already known to interact with promoter DNA and to comprise a helix-turn-helix motif. The remaining variable positions are located on previously unexplored sites inside region σ4, specifically into the first two α-helices of the region. Neither of the variable positions confined to these helices seem to interact directly with promoter sequence; instead, we adduce that these residues participate allosterically by contributing to correct region folding and/or positioning of the HTH motif. We propose that transcriptional laxity is a mechanism for ensuring transcription in spite of naturally occurring mutations from endogenous promoters and/or horizontally transferred DNA sequences, allowing survival and fast environmental adaptation of α-proteobacteria.

From indole to pyrrole, furan, thiophene and pyridine: Search for novel small molecule inhibitors of bacterial transcription initiation complex formation

The search for small molecules capable of inhibiting transcription initiation in bacteria has resulted in the synthesis of N,N′-disubstituted hydrazines and imine-carbohydrazides comprised of indole, pyridine, pyrrole, furan and thiophene using the respective trichloroacetyl derivatives, carbohydrazides and aldehydes. Replacement of the indole moiety by smaller heterocycles linked by CONHNC linkers afforded a broad variety of compounds efficiently targeting the RNA polymerase–σ70/σA interaction as determined by ELISA and exhibiting increased inhibition of the growth of Escherichia coli compared to Bacillus subtilis in culture. The structural features of the synthesized transcription initiation inhibitors needed for antibacterial activity were identified employing molecular modelling and structure–activity relationship (SAR) studies.

Elucidation of sigma factor-associated networks in Pseudomonas aeruginosa reveals a modular architecture with limited and function-specific crosstalk.

Sigma factors are essential global regulators of transcription initiation in bacteria which confer promoter recognition specificity to the RNA polymerase core enzyme. They provide effective mechanisms for simultaneously regulating expression of large numbers of genes in response to challenging conditions, and their presence has been linked to bacterial virulence and pathogenicity. In this study, we constructed nine his-tagged sigma factor expressing and/or deletion mutant strains in the opportunistic pathogen Pseudomonas aeruginosa. To uncover the direct and indirect sigma factor regulons, we performed mRNA profiling, as well as chromatin immunoprecipitation coupled to high-throughput sequencing. We furthermore elucidated the de novo binding motif of each sigma factor, and validated the RNA- and ChIP-seq results by global motif searches in the proximity of transcriptional start sites (TSS). Our integrated approach revealed a highly modular network architecture which is composed of insulated functional sigma factor modules. Analysis of the interconnectivity of the various sigma factor networks uncovered a limited, but highly function-specific, crosstalk which orchestrates complex cellular processes. Our data indicate that the modular structure of sigma factor networks enables P. aeruginosa to function adequately in its environment and at the same time is exploited to build up higher-level functions by specific interconnections that are dominated by a participation of RpoN.

Synthesis and biological activity of novel mono-indole and mono-benzofuran inhibitors of bacterial transcription initiation complex formation

Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targeted the essential protein–protein interaction between RNA polymerase core and σ70/σA factors in bacteria. In this study, the reaction of indole-2-, indole-3-, indole-7- and benzofuran-2-glyoxyloyl chlorides with amines and hydrazines afforded a variety of glyoxyloylamides and glyoxyloylhydrazides. Similarly, condensation of 2- and 7-trichloroacetylindoles with amines and hydrazines delivered amides and hydrazides. The novel molecules were found to inhibit the RNA polymerase–σ70/σA interaction as measured by ELISA, and also inhibited the growth of both Gram-positive and Gram-negative bacteria in culture. Structure–activity relationship (SAR) studies of the mono-indole and mono-benzofuran inhibitors suggested that the hydrophilic–hydrophobic balance is an important determinant of biological activity.

Remodeling of the σ 70 Subunit Non-template DNA Strand Contacts During the Final Step of Transcription Initiation

Transcription initiation in bacteria requires melting of approximately 13 bp of promoter DNA. The mechanism of the melting process is not fully understood. Escherichia coli RNA polymerase bearing a deletion of the beta subunit lobe I (amino acid residues 186-433) initiates melting of the -10 promoter element but cannot propagate the melting downstream, towards the transcription initiation start site (+1). However, in the presence of nucleotides, stable downstream melting is induced. Here, we studied lacUV5 promoter complexes formed by the mutant enzyme by cross-linking RNA polymerase subunits to single-stranded DNA in the transcription bubble. In the absence of NTPs, a contact between the sigma70 subunit and the non-template strand of the -10 promoter element was detected. This contact disappeared in the presence of NTPs. Instead, a new sigma70-DNA contact as well as stable beta' and beta subunit contacts with the non-template DNA downstream of the -10 promoter element were established. In terms of the two-step (upstream initiation/downstream propagation) model of promoter melting, our data suggest that beta lobe I induces the propagation of promoter melting by directing downstream promoter DNA duplex towards the downstream DNA-binding channel (beta' clamp). Establishment of downstream contacts leads to remodeling of upstream interactions between sigma70 and the -10 promoter element that might facilitate promoter escape and sigma release.

Activation and repression of transcription initiation in bacteria.

Transcription initiation is the principal step at which bacterial gene expression is regulated. Bacterial transcription is due to a single multisubunit RNA polymerase. The potential transcription initiation rate of any promoter is set by the efficiency with which RNA polymerase recognizes the different promoter sequence elements. The sigma subunit plays the major role in the process of promoter recognition. Different RNA polymerase sigma subunits can guide RNA polymerase to different promoters. The E. coli genome encodes seven different sigma subunits, each of which allows the cell to respond to different environmental stimuli. A large number of transcription factors up-regulate and down-regulate expression from different promoters in response to environmental signals. Many transcription activators function by making a direct interaction with RNA polymerase. Some activators function by altering the conformation of promoter DNA. Most transcription repressors function by blocking access of RNA polymerase to their target promoter. In some cases, optimal repression depends on multiply bound repressor molecules that interact in complex ways. Many promoters are regulated by more than one transcription factor. A variety of mechanisms whereby a promoter can be regulated by a repressor and an activator, or by two activators, is known.

Repression of transcription initiation in bacteria.

Bacteria live in habitats of frequently changing conditions and have evolved very sophisticated responses to adapt to environmental changes. These responses most frequently lead to the activation and/or repression of a number of genes to adapt cell physiology or metabolism to the new conditions. As

The regulation of transcription initiation in bacteria.

INTRODUCTION 355 PROMOTER ST UCTURE 356 THE REGULATION F E~TM TRANSCRIPTION I ITIATION 360 Negative Regulation of Transcription Initiation 361 Positive Regulation of Transcription Initiation 363 DNA Topology Regulation of Transcription Initiation 365 DNA Modification and the Regulation of Gene Expression 366 MODIFICATION OF HOLOENZYME STRUCTURE AND THE REGULATION OF GENE XPRESSION 366 E. coil ~r7° 367 The Heat Shock Response and ~r~2 368 The E. coli ntrA (glnF) Protein: Another Sigma Factor 370 B. subtilis Sporulation 371 Sigma Factors in B. subtilis 372 Phage-Encoded Sigma Factors 376 Conservation of Structure Among Sigma Factors 377 Modulation of Sigma Factor Use 377 Role of Sigma in Promoter Recognition 379

The Regulation of Transcription Initiation in Bacteria

The Regulation of Transcription Initiation in Bacteria

POSITIVE CONTROL OF TRANSCRIPTION INITIATION IN BACTERIA

POSITIVE CONTROL OF TRANSCRIPTION INITIATION IN BACTERIA

Positive Control of Transcription Initiation in Bacteria

Positive Control of Transcription Initiation in Bacteria