Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ70-RNAP and the λPR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.

Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAP), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, time-resolved cryo-electron microscopy (cryo-EM) was used to capture four intermediates populated 120 or 500 milliseconds (ms) after mixing Escherichia coli σ70-RNAP and the λPR promoter. Cryo-EM snapshots revealed the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As nt-strand "read-out" extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating yet unknown conformational changes load it in subsequent steps. Because these events likely describe DNA opening at many bacterial promoters, this study provides needed insights into how DNA sequence regulates steps of RPo formation.

Structural origins of Escherichia coli RNA polymerase open promoter complex stability.

The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms "open" complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λPR (t1/2 ∼10 h), T7A1 (t1/2 ∼4 min), and a point mutant in λPR (λPR-5C) (t1/2 ∼2 h). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand "scrunches" inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.

Native Mass Spectrometry-Based Screening for Optimal Sample Preparation in Single-Particle Cryo-EM.

Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well suited to routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.

Time-resolved cryo-EM using Spotiton.

We present an approach for preparing cryo-electron microscopy (cryo-EM) grids to study short-lived molecular states. Using piezoelectric dispensing, two independent streams of ~50-pl droplets of sample are deposited within 10 ms of each other onto the surface of a nanowire EM grid, and the mixing reaction stops when the grid is vitrified in liquid ethane ~100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.

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.

Transcription initiation in mycobacteria: a biophysical perspective.

Recent biophysical studies of mycobacterial transcription have shed new light on this fundamental process in a group of bacteria that includes deadly pathogens such as Mycobacterium tuberculosis (Mtb), Mycobacterium abscessus (Mab), Mycobacterium leprae (Mlp), as well as the nonpathogenic Mycobacterium smegmatis (Msm). Most of the research has focused on Mtb, the causative agent of tuberculosis (TB), which remains one of the top ten causes of death globally. The enzyme RNA polymerase (RNAP) is responsible for all bacterial transcription and is a target for one of the crucial antibiotics used for TB treatment, rifampicin (Rif). Here, we summarize recent biophysical studies of mycobacterial RNAP that have advanced our understanding of the basic process of transcription, have revealed novel paradigms for regulation, and thus have provided critical information required for developing new antibiotics against this deadly disease.

Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA.

RbpA and CarD are essential transcription regulators in mycobacteria. Mechanistic analyses of promoter open complex (RPo) formation establish that RbpA and CarD cooperatively stimulate formation of an intermediate (RP2) leading to RPo; formation of RP2 is likely a bottleneck step at the majority of mycobacterial promoters. Once RPo forms, CarD also disfavors its isomerization back to RP2. We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Both CarD and RbpA bind near the upstream edge of the -10 element where they likely facilitate DNA bending and impede transcription bubble collapse. In vivo studies demonstrate the essential role of RbpA, show the effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. This work provides a framework to understand the control of mycobacterial transcription by RbpA and CarD.

Nonspecific DNA binding and bending by HUαß: interfaces of the three binding modes characterized by salt-dependent thermodynamics.

Previous isothermal titration calorimetry (ITC) and Förster resonance energy transfer studies demonstrated that Escherichia coli HU(αß) binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34-bp mode that interacts with DNA in large (>34 bp) gaps between bound proteins, reversibly bending it by 140(o) and thereby increasing its flexibility, and two weaker, modestly cooperative small site-size modes (10 bp and 6 bp) that are useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and we deduce that DNA in the 34-bp mode is bent around-but not wrapped on-the body of HU, in contrast to specific binding of integration host factor. Analyses of binding isotherms (8-bp, 15-bp, and 34-bp DNA) and initial binding heats (34-bp, 38-bp, and 160-bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Sk(i)) even though their binding site sizes differ greatly; the most probable values of Sk(i) on 34-bp DNA or larger DNA are -7.5±0.5. From the similarity of Sk(i) values, we conclude that the binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent DNA 34-bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6-bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Sk(i) values are proposed.

Mechanism of bacterial transcription initiation: RNA polymerase - promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis.

Initiation of RNA synthesis from DNA templates by RNA polymerase (RNAP) is a multi-step process, in which initial recognition of promoter DNA by RNAP triggers a series of conformational changes in both RNAP and promoter DNA. The bacterial RNAP functions as a molecular isomerization machine, using binding free energy to remodel the initial recognition complex, placing downstream duplex DNA in the active site cleft and then separating the nontemplate and template strands in the region surrounding the start site of RNA synthesis. In this initial unstable "open" complex the template strand appears correctly positioned in the active site. Subsequently, the nontemplate strand is repositioned and a clamp is assembled on duplex DNA downstream of the open region to form the highly stable open complex, RP(o). The transcription initiation factor, σ(70), plays critical roles in promoter recognition and RP(o) formation as well as in early steps of RNA synthesis.

One-step DNA melting in the RNA polymerase cleft opens the initiation bubble to form an unstable open complex.

Though opening of the start site (+1) region of promoter DNA is required for transcription by RNA polymerase (RNAP), surprisingly little is known about how and when this occurs in the mechanism. Early events at the lambdaP(R) promoter load this region of duplex DNA into the active site cleft of Escherichia coli RNAP, forming the closed, permanganate-unreactive intermediate I(1). Conversion to the subsequent intermediate I(2) overcomes a large enthalpic barrier. Is I(2) open? Here we create a burst of I(2) by rapidly destabilizing open complexes (RP(o)) with 1.1 M NaCl. Fast footprinting reveals that thymines at positions from -11 to +2 in I(2) are permanganate-reactive, demonstrating that RNAP opens the entire initiation bubble in the cleft in a single step. Rates of decay of all observed thymine reactivities are the same as the I(2) to I(1) conversion rate determined by filter binding. In I(2), permanganate reactivity of the +1 thymine on the template (t) strand is the same as the RP(o) control, whereas nontemplate (nt) thymines are significantly less reactive than in RP(o). We propose that: (i) the +1(t) thymine is in the active site in I(2); (ii) conversion of I(2) to RP(o) repositions the nt strand in the cleft; and (iii) movements of the nt strand are coupled to the assembly and DNA binding of the downstream clamp and jaw that occurs after DNA opening and stabilizes RP(o). We hypothesize that unstable open intermediates at the lambdaP(R) promoter resemble the unstable, transcriptionally competent open complexes formed at ribosomal promoters.

Probing DNA binding, DNA opening, and assembly of a downstream clamp/jaw in Escherichia coli RNA polymerase-lambdaP(R) promoter complexes using salt and the physiological anion glutamate.

Transcription by all RNA polymerases (RNAPs) requires a series of large-scale conformational changes to form the transcriptionally competent open complex RP(o). At the lambdaP(R) promoter, Escherichia coli sigma(70) RNAP first forms a wrapped, closed 100 bp complex I(1). The subsequent step opens the entire DNA bubble, creating the relatively unstable (open) complex I(2). Additional conformational changes convert I(2) to the stable RP(o). Here we probe these events by dissecting the effects of Na(+) salts of Glu(-), F(-), and Cl(-) on each step in this critical process. Rapid mixing and nitrocellulose filter binding reveal that the binding constant for I(1) at 25 degrees C is approximately 30-fold larger in Glu(-) than in Cl(-) at the same Na(+) concentration, with the same log-log salt concentration dependence for both anions. In contrast, both the rate constant and equilibrium constant for DNA opening (I(1) to I(2)) are only weakly dependent on salt concentration, and the opening rate constant is insensitive to replacement of Cl(-) with Glu(-). These very small effects of salt concentration on a process (DNA opening) that is strongly dependent on salt concentration in solution may indicate that the backbones of both DNA strands interact with polymerase throughout the process and/or that compensation is present between ion uptake and release. Replacement of Cl(-) with Glu(-) or F(-) at 25 degrees C greatly increases the lifetime of RP(o) and greatly reduces its salt concentration dependence. By analogy to Hofmeister salt effects on protein folding, we propose that the excluded anions Glu(-) and F(-) drive the folding and assembly of the RNAP clamp/jaw domains in the conversion of I(2) to RP(o), while Cl(-) does not. Because the Hofmeister effect of Glu(-) or F(-) largely compensates for the destabilizing Coulombic effect of any salt on the binding of this assembly to downstream promoter DNA, RP(o) remains long-lived even at 0.5 M Na(+) in Glu(-) or F(-) salts. The observation that Esigma(70) RP(o) complexes are exceedingly long-lived at moderate to high Glu(-) concentrations argues that Esigma(70) RNAP does not dissociate from strong promoters in vivo when the cytoplasmic glutamate concentration increases during osmotic stress.

Interactions of the osmolyte glycine betaine with molecular surfaces in water: thermodynamics, structural interpretation, and prediction of m-values.

Noncovalent self-assembly of biopolymers is driven by molecular interactions between functional groups on complementary biopolymer surfaces, replacing interactions with water. Since individually these interactions are comparable in strength to interactions with water, they have been difficult to quantify. Solutes (osmolytes, denaturants) exert often large effects on these self-assembly interactions, determined in sign and magnitude by how well the solute competes with water to interact with the relevant biopolymer surfaces. Here, an osmometric method and a water-accessible surface area (ASA) analysis are developed to quantify and interpret the interactions of the remarkable osmolyte glycine betaine (GB) with molecular surfaces in water. We find that GB, lacking hydrogen bond donors, is unable to compete with water to interact with anionic and amide oxygens; this explains its effectiveness as an osmolyte in the Escherichia coli cytoplasm. GB competes effectively with water to interact with amide and cationic nitrogens (hydrogen bonding) and especially with aromatic hydrocarbon (cation-pi). The large stabilizing effect of GB on lac repressor-lac operator binding is predicted quantitatively from ASA information and shown to result largely from dehydration of anionic DNA phosphate oxygens in the protein-DNA interface. The incorporation of these results into theoretical and computational analyses will likely improve the ability to accurately model intra- and interprotein interactions. Additionally, these results pave the way for development of solutes as kinetic/mechanistic and thermodynamic probes of conformational changes and formation/disruption of molecular interfaces that occur in the steps of biomolecular self-assembly processes.

Evidence for a tyrosine-adenine stacking interaction and for a short-lived open intermediate subsequent to initial binding of Escherichia coli RNA polymerase to promoter DNA.

Bacterial RNA polymerase and a "sigma" transcription factor form an initiation-competent "open" complex at a promoter by disruption of about 14 base pairs. Strand separation is likely initiated at the highly conserved -11 A-T base pair. Amino acids in conserved region 2.3 of the main Escherichia coli sigma factor, sigma(70), are involved in this process, but their roles are unclear. To monitor the fates of particular bases upon addition of RNA polymerase, promoters bearing single substitutions of the fluorescent A-analog 2-aminopurine (2-AP) at -11 and two other positions in promoter DNA were examined. Evidence was obtained for an open intermediate on the pathway to open complex formation, in which these 2-APs are no longer stacked onto their neighboring bases. The tyrosine at residue 430 in region 2.3 of sigma(70) was shown to be involved in quenching the fluorescence of a 2-AP substituted at -11, presumably through a stacking interaction. These data refine the structural model for open complex formation and reveal a novel interaction involved in DNA melting by RNA polymerase.

DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA--protein complex on intact, linear duplex DNA.

Escherichia coli HU(alphabeta), a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HU(alphabeta) interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (> or =34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1+/-0.4x10(6) M(-1), and a binding enthalpy of +7.7+/-0.6 kcal/mol at 15 degrees C and 0.15 M Na(+). With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (omega approximately 20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1+/-0.2x10(5) M(-1) for the 10 bp mode, 3.5+/-1.4x10(4) M(-1) for the 6 bp mode) and binding enthalpies (4.2+/-0.3 kcal/mol for the 10 bp mode, -1.6+/-0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143+/-6 degrees whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA.

Formation of a wrapped DNA-protein interface: experimental characterization and analysis of the large contributions of ions and water to the thermodynamics of binding IHF to H' DNA.

To characterize driving forces and driven processes in formation of a large-interface, wrapped protein-DNA complex analogous to the nucleosome, we have investigated the thermodynamics of binding the 34-base pair (bp) H' DNA sequence to the Escherichia coli DNA-remodeling protein integration host factor (IHF). Isothermal titration calorimetry and fluorescence resonance energy transfer are applied to determine effects of salt concentration [KCl, KF, K glutamate (KGlu)] and of the excluded solute glycine betaine (GB) on the binding thermodynamics at 20 degrees C. Both the binding constant K(obs) and enthalpy Delta H degrees (obs) depend strongly on [salt] and anion identity. Formation of the wrapped complex is enthalpy driven, especially at low [salt] (e.g., Delta H(o)(obs)=-20.2 kcal x mol(-1) in 0.04 M KCl). Delta H degrees (obs) increases linearly with [salt] with a slope (d Delta H degrees (obs)/d[salt]), which is much larger in KCl (38+/-3 kcal x mol(-1) M(-1)) than in KF or KGlu (11+/-2 kcal x mol(-1) M(-1)). At 0.33 M [salt], K(obs) is approximately 30-fold larger in KGlu or KF than in KCl, and the [salt] derivative SK(obs)=dlnK(obs)/dln[salt] is almost twice as large in magnitude in KCl (-8.8+/-0.7) as in KF or KGlu (-4.7+/-0.6). A novel analysis of the large effects of anion identity on K(obs), SK(obs) and on Delta H degrees (obs) dissects coulombic, Hofmeister, and osmotic contributions to these quantities. This analysis attributes anion-specific differences in K(obs), SK(obs), and Delta H degrees (obs) to (i) displacement of a large number of water molecules of hydration [estimated to be 1.0(+/-0.2)x10(3)] from the 5340 A(2) of IHF and H' DNA surface buried in complex formation, and (ii) significant local exclusion of F(-) and Glu(-) from this hydration water, relative to the situation with Cl(-), which we propose is randomly distributed. To quantify net water release from anionic surface (22% of the surface buried in complexation, mostly from DNA phosphates), we determined the stabilizing effect of GB on K(obs): dlnK(obs)/d[GB]=2.7+/-0.4 at constant KCl activity, indicating the net release of ca. 150 H(2)O molecules from anionic surface.

Late steps in the formation of E. coli RNA polymerase-lambda P R promoter open complexes: characterization of conformational changes by rapid [perturbant] upshift experiments.

The formation of the transcriptionally competent open complex (RP(o)) by Escherichia coli RNA polymerase at the lambda P(R) promoter involves at least three steps and two kinetically significant intermediates (I(1) and I(2)). Understanding the sequence of conformational changes (rearrangements in the jaws of RNA polymerase, DNA opening) that occur in the conversion of I(1) to RP(o) requires: (1) dissecting the rate constant k(d) for the dissociation of RP(o) into contributions from individual steps and (2) isolating and characterizing I(2). To deconvolute k(d), we develop experiments involving rapid upshifts to elevated concentrations of RP(o)-destabilizing solutes ("perturbants": urea and KCl) to create a burst in the population of I(2). At high concentrations of either perturbant, k(d) approaches the same [perturbant]-independent value, interpreted as the elementary rate constant k(-2) for I(2)-->I(1). The large effects of [urea] and [salt] on K(3) (the equilibrium constant for I(2) is in equilibrium with RP(o)) indicate that a large-scale folding transition in polymerase occurs and a new interface with the DNA forms late in the mechanism. We deduce that I(2) at the lambda P(R) promoter is always unstable relative to RP(o), even at 0 degrees C, explaining previous difficulties in detecting it by using temperature downshifts. The division of the large positive enthalpy change between the late steps of the mechanism suggests that an additional unstable intermediate (I(3)) may exist between I(2) and RP(o).

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase.

The architecture of cellular RNA polymerases (RNAPs) dictates that transcription can begin only after promoter DNA bends into a deep channel and the start site nucleotide (+1) binds in the active site located on the channel floor. Formation of this transcriptionally competent "open" complex (RP(o)) by Escherichia coli RNAP at the lambdaP(R) promoter is greatly accelerated by DNA upstream of base pair -47 (with respect to +1). Here we report real-time hydroxyl radical (*OH) and potassium permanganate (KMnO4) footprints obtained under conditions selected for optimal characterization of the first kinetically significant intermediate (I(1)) in RP(o) formation. .OH footprints reveal that the DNA backbone from -71 to -81 is engulfed by RNAP in I(1) but not in RP(o); downstream protection extends to approximately +20 in both complexes. KMnO4 footprinting detects solvent-accessible thymine bases in RP(o), but not in I(1). We conclude that upstream DNA wraps more extensively on RNAP in I(1) than in RP(o) and that downstream DNA (-11 to +20) occupies the active-site channel in I(1) but is not yet melted. Mapping of the footprinting data onto available x-ray structures provides a detailed model of a kinetic intermediate in bacterial transcription initiation and suggests how transient contacts with upstream DNA in I(1) might rearrange the channel to favor entry of downstream duplex DNA.

Synthesis-mediated release of a small RNA inhibitor of RNA polymerase.

Noncoding small RNAs regulate gene expression in all organisms, in some cases through direct association with RNA polymerase (RNAP). Here we report that the mechanism of 6S RNA inhibition of transcription is through specific, stable interactions with the active site of Escherichia coli RNAP that exclude promoter DNA binding. In fact, the DNA-dependent RNAP uses bound 6S RNA as a template for RNA synthesis, producing 14-to 20-nucleotide RNA products (pRNA). These results demonstrate that 6S RNA is functionally engaged in the active site of RNAP. Synthesis of pRNA destabilizes 6S RNA-RNAP complexes leading to release of the pRNA-6S RNA hybrid. In vivo, 6S RNA-directed RNA synthesis occurs during outgrowth from the stationary phase and likely is responsible for liberating RNAP from 6S RNA in response to nutrient availability.

Do sigma factors need help with a meltdown?

In this issue of Cell, Hsu et al. (2006) report on the binding activity of a variant of the bacterial transcriptional specificity factor sigma (sigma) to promoter DNA. This study demonstrates that the sigma variant induces a large distortion in the transcriptional start site in the absence of core RNA polymerase, raising intriguing new questions about the roles of sigma and core RNA polymerase in transcription initiation.