Identification and characterization of RNA binding sites for (p)ppGpp using RNA-DRaCALA.

Ligand-binding RNAs (RNA aptamers) are widespread in the three domains of life, serving as sensors of metabolites and other small molecules. When aptamers are embedded within RNA transcripts as components of riboswitches, they can regulate gene expression upon binding their ligands. Previous methods for biochemical validation of computationally predicted aptamers are not well-suited for rapid screening of large numbers of RNA aptamers.

Homologs of the Escherichia coli F Element Protein TraR, Including Phage Lambda Orf73, Directly Reprogram Host Transcription.

Bacterial cells and their associated plasmids and bacteriophages encode numerous small proteins of unknown function. One example, the 73-amino-acid protein TraR, is encoded by the transfer operon of the conjugative F plasmid of Escherichia coli. TraR is a distant homolog of DksA, a protein found in almost all proteobacterial species that is required for ppGpp to regulate transcription during the stringent response.

Rhodobacter sphaeroides CarD Negatively Regulates Its Own Promoter.

Bioinformatic analysis showed previously that a majority of promoters in the photoheterotrophic alphaproteobacterium Rhodobacter sphaeroides lack the thymine at the last position of the -10 element (-7T), a base that is very highly conserved in promoters in bacteria other than alphaproteobacteria. The absence of -7T was correlated with low promoter activity using purified R. sphaeroides RNA polymerase (RNAP), but the transcription factor CarD compensated by activating almost all promoters lacking -7T tested , including rRNA promoters.

A majority of promoters lack a crucial RNA polymerase recognition feature, enabling coordinated transcription activation.

Using an in vitro transcription system with purified RNA polymerase (RNAP) to investigate rRNA synthesis in the photoheterotrophic α-proteobacterium , we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription from rRNA promoters was unexpectedly weak, correlating with absence of -7T, the very highly conserved thymine found at the last position in -10 elements of promoters in most bacterial species. Thymine substitutions for adenine at position -7 in the three rRNA promoters strongly increased intrinsic promoter activity, indicating that RNAP can utilize -7T when present.

Reverse engineering of fatty acid-tolerant Escherichia coli identifies design strategies for robust microbial cell factories.

Adaptive laboratory evolution is often used to improve the performance of microbial cell factories. Reverse engineering of evolved strains enables learning and subsequent incorporation of novel design strategies via the design-build-test-learn cycle. Here, we reverse engineer a strain of Escherichia coli previously evolved for increased tolerance of octanoic acid (C8), an attractive biorenewable chemical, resulting in increased C8 production, increased butanol tolerance, and altered membrane properties.

Stepwise Promoter Melting by Bacterial RNA Polymerase.

Transcription initiation requires formation of the open promoter complex (RPo). To generate RPo, RNA polymerase (RNAP) unwinds the DNA duplex to form the transcription bubble and loads the DNA into the RNAP active site. RPo formation is a multi-step process with transient intermediates of unknown structure.

TraR allosterically regulates transcription initiation by altering RNA polymerase conformation.

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of RNAP, with or without TraR, and of an RNAP-promoter complex.

Structural basis for transcription activation by Crl through tethering of σ and RNA polymerase.

In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ-factors are negatively regulated by anti-σ-factors. In , , and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σ-regulon by promoting the association of σ with RNAP without interacting with promoter DNA.

Genome-wide effects on transcription from ppGpp binding to its two sites on RNA polymerase.

The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) in , but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP.

Transcriptional Responses to ppGpp and DksA.

The stringent response to nutrient deprivation is a stress response found throughout the bacterial domain of life. Although first described in proteobacteria for matching ribosome synthesis to the cell's translation status and for preventing formation of defective ribosomal particles, the response is actually much broader, regulating many hundreds of genes-some positively, some negatively. Utilization of the signaling molecules ppGpp and pppGpp for this purpose is ubiquitous in bacterial evolution, although the mechanisms employed vary.

DksA and ppGpp Regulate the σ Stress Response by Activating Promoters for the Small RNA DsrA and the Anti-Adapter Protein IraP.

σ is an alternative sigma factor, encoded by the gene, that redirects cellular transcription to a large family of genes in response to stressful environmental signals. This so-called σ general stress response is necessary for survival in many bacterial species and is controlled by a complex, multifactorial pathway that regulates σ levels transcriptionally, translationally, and posttranslationally in It was shown previously that the transcription factor DksA and its cofactor, ppGpp, are among the many factors governing σ synthesis, thus playing an important role in activation of the σ stress response. However, the mechanisms responsible for the effects of DksA and ppGpp have not been elucidated fully.

Dissection of the molecular circuitry controlling virulence in .

the etiological agent of tularemia, is one of the most infectious bacteria known. Because of its extreme pathogenicity, is classified as a category A bioweapon by the US government. virulence stems from genes encoded on the pathogenicity island (FPI).

Roles of Transcriptional and Translational Control Mechanisms in Regulation of Ribosomal Protein Synthesis in Escherichia coli.

Bacterial ribosome biogenesis is tightly regulated to match nutritional conditions and to prevent formation of defective ribosomal particles. In , most ribosomal protein (r-protein) synthesis is coordinated with rRNA synthesis by a translational feedback mechanism: when r-proteins exceed rRNAs, specific r-proteins bind to their own mRNAs and inhibit expression of the operon. It was recently discovered that the second messenger nucleotide guanosine tetra and pentaphosphate (ppGpp), which directly regulates rRNA promoters, is also capable of regulating many r-protein promoters.

TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp.

The F element-encoded protein TraR is a distant homolog of the chromosome-encoded transcription factor DksA. Here we address the mechanism by which TraR acts as a global regulator, inhibiting some promoters and activating others. We show that TraR regulates transcription directly in vitro by binding to the secondary channel of RNA polymerase (RNAP) using interactions similar, but not identical, to those of DksA.

Open complex DNA scrunching: A key to transcription start site selection and promoter escape.

Bacterial RNA polymerase-promoter open complexes can exist in a range of states in which the leading edge of the enzyme moves but the trailing edge does not, a phenomenon we refer to as "open complex scrunching." Here we describe how open complex scrunching can determine the position of the transcription start site for some promoters, modulate the level of expression, and potentially could be targeted by factors to regulate transcription. We suggest that open complex scrunching at the extraordinarily active ribosomal RNA promoters might have evolved to initiate transcription at an unusual position relative to the core promoter elements in order to maximize the rate of promoter escape.

Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus.

The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chromosome organization in Escherichia coli, we examined the relative positions of the ribosomal RNA (rRNA) operons in space. The seven rRNA operons are nearly identical and separated from each other by as much as 180° on the circular genetic map, a distance of ≥2 million base pairs.

ppGpp Binding to a Site at the RNAP-DksA Interface Accounts for Its Dramatic Effects on Transcription Initiation during the Stringent Response.

Throughout the bacterial domain, the alarmone ppGpp dramatically reprograms transcription following nutrient limitation. This "stringent response" is critical for survival and antibiotic tolerance and is a model for transcriptional regulation by small ligands. We report that ppGpp binds to two distinct sites 60 Å apart on E.

Coexpression of Escherichia coli obgE, Encoding the Evolutionarily Conserved Obg GTPase, with Ribosomal Proteins L21 and L27.

Unlabelled: Multiple essential small GTPases are involved in the assembly of the ribosome or in the control of its activity. Among them, ObgE (CgtA) has been shown recently to act as a ribosome antiassociation factor that binds to ppGpp, a regulator whose best-known target is RNA polymerase. The present study was aimed at elucidating the expression of obgE in Escherichia coli We show that obgE is cotranscribed with ribosomal protein genes rplU and rpmA and with a gene of unknown function, yhbE We show here that about 75% of the transcripts terminate before obgE, because there is a transcriptional terminator between rpmA and yhbE As expected for ribosomal protein operons, expression was highest during exponential growth, decreased during entry into stationary phase, and became almost undetectable thereafter.