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. We use single-particle cryoelectron microscopy to visualize seven intermediates containing Escherichia coli RNAP with the transcription factor TraR en route to forming RPo. The structures span the RPo formation pathway from initial recognition of the duplex promoter in a closed complex to the final RPo. The structures and supporting biochemical data define RNAP and promoter DNA conformational changes that delineate steps on the pathway, including previously undetected transient promoter-RNAP interactions that contribute to populating the intermediates but do not occur in RPo. Our work provides a structural basis for understanding RPo formation and its regulation, a major checkpoint in gene expression throughout evolution.

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. Therefore, we utilized DRaCALA (Differential Radial Capillary Action of Ligand Assay), a technique designed originally to study protein-ligand interactions, to examine RNA-ligand binding, permitting rapid screening of dozens of RNA aptamer candidates concurrently. Using this method, which we call RNA-DRaCALA, we screened 30 ykkC family subtype 2a RNA aptamers that were computationally predicted to bind (p)ppGpp. Most of the aptamers bound both ppGpp and pppGpp, but some strongly favored only ppGpp or pppGpp, and some bound neither. Expansion of the number of biochemically verified sites allowed construction of more accurate secondary structure models and prediction of key features in the aptamers that distinguish a ppGpp from a pppGpp binding site. To demonstrate that the method works with other ligands, we also used RNA DRaCALA to analyze aptamer binding by thiamine pyrophosphate.

Dissection of the molecular circuitry controlling virulence in Francisella tularensis.

Francisella tularensis, the etiological agent of tularemia, is one of the most infectious bacteria known. Because of its extreme pathogenicity, F. tularensis is classified as a category A bioweapon by the US government. F. tularensis virulence stems from genes encoded on the Francisella pathogenicity island (FPI). An unusual set of Francisella regulators-the heteromeric macrophage growth locus protein A (MglA)-stringent starvation protein A (SspA) complex and the DNA-binding protein pathogenicity island gene regulator (PigR)-activates FPI transcription and thus is essential for virulence. Intriguingly, the second messenger, guanosine-tetraphosphate (ppGpp), which is produced during infection, is also involved in coordinating Francisella virulence; however, its role has been unclear. Here we identify MglA-SspA as a novel ppGpp-binding complex and describe structures of apo- and ppGpp-bound MglA-SspA. We demonstrate that MglA-SspA, which binds RNA polymerase (RNAP), also interacts with the C-terminal domain of PigR, thus anchoring the (MglA-SspA)-RNAP complex to the FPI promoter. Furthermore, we show that MglA-SspA must be bound to ppGpp to mediate high-affinity interactions with PigR. Thus, these studies unveil a novel pathway different from those described previously for regulation of transcription by ppGpp. The data also indicate that F. tularensis pathogenesis is controlled by a highly interconnected molecular circuitry in which the virulence machinery directly senses infection via a small molecule stress signal.

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. coli RNA polymerase (RNAP), one characterized previously (site 1) and a second identified here at an interface of RNAP and the transcription factor DksA (site 2). The location and unusual tripartite nature of site 2 account for the DksA-ppGpp synergism and suggest mechanisms for ppGpp enhancement of DksA's effects on RNAP. Site 2 binding results in the majority of ppGpp's effects on transcription initiation in vitro and in vivo, and strains lacking site 2 are severely impaired for growth following nutritional shifts. Filling of the two sites at different ppGpp concentrations would expand the dynamic range of cellular responses to changes in ppGpp levels.

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. In proteobacteria, the signaling molecules typically bind to two sites on RNA polymerase, one at the interface of the ß' and ω subunits and one at the interface of the ß' secondary channel and the transcription factor DksA. The ß' secondary channel is targeted by other transcription regulators as well. Although studies on the transcriptional outputs of the stringent response date back at least 50 years, the mechanisms responsible are only now coming into focus.

Crosslink Mapping at Amino Acid-Base Resolution Reveals the Path of Scrunched DNA in Initial Transcribing Complexes.

RNA polymerase binds tightly to DNA to recognize promoters with high specificity but then releases these contacts during the initial stage of transcription. We report a site-specific crosslinking approach to map the DNA path in bacterial transcription intermediates at amino acid and nucleotide resolution. After validating the approach by showing that the DNA path in open complexes (RPO) is the same as in high-resolution X-ray structures, we define the path following substrate addition in "scrunched" complexes (RPITC). The DNA bulges that form within the transcription bubble in RPITC are positioned differently on the two strands. Our data suggest that the non-template strand bulge is extruded into solvent in complexes containing a 5-mer RNA, whereas the template strand bulge remains within the template strand tunnel, exerting stress on interactions between the ß flap, ß' clamp, and σ3.2. We propose that this stress contributes to σ3.2 displacement from the RNA exit channel, facilitating promoter escape.

A majority of Rhodobacter sphaeroides 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 Rhodobacter sphaeroides, we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription from R. sphaeroides 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 R. sphaeroides RNAP can utilize -7T when present. rRNA promoters were activated by purified R. sphaeroides CarD, a transcription factor found in many bacterial species but not in ß- and γ-proteobacteria. Overall, CarD increased the activity of 15 of 16 native R. sphaeroides promoters tested in vitro that lacked -7T, whereas it had no effect on three of the four native promoters that contained -7T. Genome-wide bioinformatic analysis of promoters from R. sphaeroides and two other α-proteobacterial species indicated that 30 to 43% contained -7T, whereas 90 to 99% of promoters from non-α-proteobacteria contained -7T. Thus, promoters lacking -7T appear to be widespread in α-proteobacteria and may have evolved away from consensus to enable their coordinated regulation by transcription factors like CarD. We observed a strong reduction in R. sphaeroides CarD levels when cells enter stationary phase, suggesting that reduced activation by CarD may contribute to inhibition of rRNA transcription when cells enter stationary phase, the stage of growth when bacterial ribosome synthesis declines.

Genome-wide effects on Escherichia coli 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 Escherichia coli, 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. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.

Structural basis for transcription activation by Crl through tethering of σS 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 Escherichia coli, Salmonella enterica, and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σS-regulon by promoting the association of σS with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σS-RNAP in an open promoter complex with a σS-regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σS (σS2), the structure, along with p-benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP ß'-subunit that we call the ß'-clamp-toe (ß'CT). Deletion of the ß'CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-ß'CT interaction. We conclude that Crl activates σS-dependent transcription in part through stabilizing σS-RNAP by tethering σS2 and the ß'CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.

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 in vitro, including rRNA promoters. Here, we show that a previously uncharacterized R. sphaeroides promoter, the promoter for carD itself, has high basal activity relative to other tested R. sphaeroides promoters despite lacking -7T, and its activity is inhibited rather than activated by CarD. This high basal activity is dependent on a consensus-extended -10 element (TGn) and specific features in the spacer immediately upstream of the extended -10 element. CarD negatively autoregulates its own promoter by producing abortive transcripts, limiting promoter escape, and reducing full-length mRNA synthesis. This mechanism of negative regulation differs from that employed by classical repressors, in which the transcription factor competes with RNA polymerase for binding to the promoter, and with the mechanism of negative regulation used by transcription factors like DksA/ppGpp and TraR that allosterically inhibit the rate of open complex formation. IMPORTANCE R. sphaeroides CarD activates many promoters by binding directly to RNAP and DNA just upstream of the -10 element. In contrast, we show here that CarD inhibits its own promoter using the same interactions with RNAP and DNA used for activation. Inhibition results from increasing abortive transcript formation, thereby decreasing promoter escape and full-length RNA synthesis. We propose that the combined interactions of RNAP with CarD, with the extended -10 element and with features in the adjacent -10/-35 spacer DNA, stabilize the promoter complex, reducing promoter clearance. These findings support previous predictions that the effects of CarD on transcription can be either positive or negative, depending on the kinetic properties of the specific promoter.

The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation.

The global regulatory nucleotide ppGpp ("magic spot") regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp's target site on RNAP, and therefore its mechanism of action, has remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and ß', and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.

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. Here, evolution was determined to have occurred first through the restoration of WaaG activity, involved in the production of lipopolysaccharides, then an amino acid change in RpoC, a subunit of RNA polymerase, and finally mutation of the BasS-BasR two component system. All three mutations were required in order to reproduce the increased growth rate in the presence of 20 mM C8 and increased cell surface hydrophobicity; the WaaG and RpoC mutations both contributed to increased C8 titers, with the RpoC mutation appearing to be the major driver of this effect. Each of these mutations contributed to changes in the cell membrane. Increased membrane integrity and rigidity and decreased abundance of extracellular polymeric substances can be attributed to the restoration of WaaG. The increase in average lipid tail length can be attributed to the RpoCH419P mutation, which also confers tolerance to other industrially-relevant inhibitors, such as furfural, vanillin and n-butanol. The RpoCH419P mutation may impact binding or function of the stringent response alarmone ppGpp to RpoC site 1. Each of these mutations provides novel strategies for engineering microbial robustness, particularly at the level of the microbial cell membrane.

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

The Escherichia coli 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. Even though it binds to RNAP with only slightly higher affinity than DksA and is only half the size of DksA, TraR by itself inhibits transcription as strongly as DksA and ppGpp combined and much more than DksA alone. Furthermore, unlike DksA, TraR activates transcription even in the absence of ppGpp. TraR lacks the residues that interact with ppGpp in DksA, and TraR binding to RNAP uses the residues in the ß' rim helices that contribute to the ppGpp binding site in the DksA-ppGpp-RNAP complex. Thus, unlike DksA, TraR does not bind ppGpp. We propose a model in which TraR mimics the effects of DksA and ppGpp together by binding directly to the region of the RNAP secondary channel that otherwise binds ppGpp, and its N-terminal region, like the coiled-coil tip of DksA, engages the active-site region of the enzyme and affects transcription allosterically. These data provide insights into the function not only of TraR but also of an evolutionarily widespread and diverse family of TraR-like proteins encoded by bacteria, as well as bacteriophages and other extrachromosomal elements.

Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation.

Escherichia coli DksA is a transcription factor that binds to RNA polymerase (RNAP) without binding to DNA, destabilizing RNAP-promoter interactions, sensitizing RNAP to the global regulator ppGpp, and regulating transcription of several hundred target genes, including those encoding rRNA. Previously, we described promoter sequences and kinetic properties that account for DksA's promoter specificity, but how DksA exerts its effects on RNAP has remained unclear. To better understand DksA's mechanism of action, we incorporated benzoyl-phenylalanine at specific positions in DksA and mapped its cross-links to RNAP, constraining computational docking of the two proteins. The resulting evidence-based model of the DksA-RNAP complex as well as additional genetic and biochemical approaches confirmed that DksA binds to the RNAP secondary channel, defined the orientation of DksA in the channel, and predicted a network of DksA interactions with RNAP that includes the rim helices and the mobile trigger loop (TL) domain. Engineered cysteine substitutions in the TL and DksA coiled-coil tip generated a disulfide bond between them, and the interacting residues were absolutely required for DksA function. We suggest that DksA traps the TL in a conformation that destabilizes promoter complexes, an interaction explaining the requirement for the DksA tip and its effects on transcription.

Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters.

Most Escherichia coli promoters initiate transcription with a purine 7 or 8 nt downstream from the -10 hexamer, but some promoters, including the ribosomal RNA promoter rrnB P1, start 9 nt from the -10 element. We identified promoter and RNA polymerase determinants of this noncanonical rrnB P1 start site using biochemical and genetic approaches including mutational analysis of the promoter, Fe(2+) cleavage assays to monitor template strand positions near the active-site, and Bpa cross-linking to map the path of open complex DNA at amino acid and nucleotide resolution. We find that mutations in several promoter regions affect transcription start site (TSS) selection. In particular, we show that the absence of strong interactions between the discriminator region and σ region 1.2 and between the extended -10 element and σ region 3.0, identified previously as a determinant of proper regulation of rRNA promoters, is also required for the unusual TSS. We find that the DNA in the single-stranded transcription bubble of the rrnB P1 promoter complex expands and is "scrunched" into the active site channel of RNA polymerase, similar to the situation in initial transcribing complexes. However, in the rrnB P1 open complex, scrunching occurs before RNA synthesis begins. We find that the scrunched open complex exhibits reduced abortive product synthesis, suggesting that scrunching and unusual TSS selection contribute to the extraordinary transcriptional activity of rRNA promoters by increasing promoter escape, helping to offset the reduction in promoter activity that would result from the weak interactions with σ.

Allosteric control of Escherichia coli rRNA promoter complexes by DksA.

The Escherichia coli DksA protein inserts into the RNA polymerase (RNAP) secondary channel, modifying the transcription initiation complex so that promoters with specific kinetic characteristics are regulated by changes in the concentrations of ppGpp and NTPs. We used footprinting assays to determine the specific kinetic intermediate, RP(I), on which DksA acts. Genetic approaches identified substitutions in the RNAP switch regions, bridge helix, and trigger loop that mimicked, reduced, or enhanced DksA function on rRNA promoters. Our results indicate that DksA binding in the secondary channel of RP(I) disrupts interactions with promoter DNA at least 25 A away, between positions -6 and +6 (the transcription start site is +1). We propose a working model in which the trigger loop and bridge helix transmit effects of DksA to the switch region(s), allosterically affecting switch residues that control clamp opening/closing and/or that interact directly with promoter DNA. DksA thus inhibits the transition to RP(I). Our results illustrate in mechanistic terms how transcription factors can regulate initiation promoter-specifically without interacting directly with DNA.

Activation of the σE-dependent stress pathway by conjugative TraR may anticipate conjugational stress.

Horizontal gene transfer by conjugation plays a major role in bacterial evolution, allowing the acquisition of new traits, such as virulence and resistance to antibacterial agents. With the increased antibiotic resistance in bacterial pathogens, a better understanding of how bacteria modulate conjugation under changing environments and the genetic factors involved is needed. Despite the evolutionary advantages conjugation may confer, the process can be quite stressful for the donor cell. Here, we characterize the ability of TraR, encoded on the episomal F' plasmid, to upregulate the σ(E) extracytoplasmic stress pathway in Escherichia coli. TraR, a DksA homolog, modulates transcription initiation through the secondary channel of RNA polymerase. We show here that TraR activates transcription directly; however, unlike DksA, it does so without using ppGpp as a cofactor. TraR expression can stimulate the σ(E) extracytoplasmic stress response independently of the DegS/RseA signal transduction cascade. In the absence of TraR, bacteria carrying conjugative plasmids become more susceptible to external stress. We propose that TraR increases the concentrations of periplasmic chaperones and proteases by directly activating the transcription of σ(E)-dependent promoters; this increased protein folding capacity may prepare the bacterium to endure the periplasmic stress of sex pilus biosynthesis during mating.

Structure of the RNA polymerase assembly factor Crl and identification of its interaction surface with sigma S.

Bacteria utilize multiple sigma factors that associate with core RNA polymerase (RNAP) to control transcription in response to changes in environmental conditions. In Escherichia coli and Salmonella enterica, Crl positively regulates the σ(S) regulon by binding to σ(S) to promote its association with core RNAP. We recently characterized the determinants in σ(S) responsible for specific binding to Crl. However, little is known about the determinants in Crl required for this interaction. Here, we present the X-ray crystal structure of a Crl homolog from Proteus mirabilis in conjunction with in vivo and in vitro approaches that probe the Crl-σ(S) interaction in E. coli. We show that the P. mirabilis, Vibrio harveyi, and E. coli Crl homologs function similarly in E. coli, indicating that Crl structure and function are likely conserved throughout gammaproteobacteria. We utilize phylogenetic conservation and bacterial two-hybrid analyses to predict residues in Crl important for the interaction with σ(S). The results of p-benzoylphenylalanine (BPA)-mediated UV cross-linking studies further support the model in which an evolutionarily conserved central cleft is the surface on Crl that binds to σ(S). Within this conserved binding surface, we identify a key residue in Crl that is critical for activation of Eσ(S)-dependent transcription in vivo and in vitro. Our study provides a physical basis for understanding the σ(S)-Crl interaction.

Direct regulation of Escherichia coli ribosomal protein promoters by the transcription factors ppGpp and DksA.

We show here that the promoters for many of the Escherichia coli ribosomal protein operons are regulated directly by two transcription factors, the small RNA polymerase-binding protein DksA and the nutritional stress-induced nucleotide ppGpp. ppGpp and DksA work together to inhibit transcription initiation from ribosomal protein promoters in vitro and in vivo. The degree of promoter regulation by ppGpp/DksA varies among the r-protein promoters, but some are inhibited almost as much as rRNA promoters. Thus, many r-protein operons are regulated at the level of transcription in addition to their control by the classic translational feedback systems discovered ~30 y ago. We conclude that direct control of r-protein promoters and rRNA promoters by the same signal, ppGpp/DksA, makes a major contribution to the balanced and coordinated synthesis rates of all of the ribosomal components.

Super DksAs: substitutions in DksA enhancing its effects on transcription initiation.

At specific times during bacterial growth, the transcription factor DksA and the unusual nucleotide regulator ppGpp work synergistically to inhibit some Escherichia coli promoters (e.g. rRNA promoters) and to stimulate others (e.g. promoters for amino-acid synthesis and transport). However, the mechanism of DksA action remains uncertain, in part because DksA does not function like conventional transcription factors. To gain insights into DksA function, we identified mutations in dksA that bypassed the requirement for ppGpp by selecting for growth of cells lacking ppGpp on minimal medium without amino acids. We show here that two substitutions in DksA, L15F and N88I, result in higher DksA activity both in vivo and in vitro, primarily by increasing the apparent affinity of DksA for RNA polymerase (RNAP). The mutant DksA proteins suggest potential roles for ppGpp in DksA function, identify potential surfaces on DksA crucial for RNAP binding, and provide tools for future studies to elucidate the mechanism of DksA action.