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In many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), the sigma factor RpoS/σ(S) accumulates during stationary phase of growth, and associates with the core RNA polymerase enzyme (E) to promote transcription initiation of genes involved in general stress resistance and starvation survival. Whereas σ factors are usually inactivated upon interaction with anti-σ proteins, σ(S) binding to the Crl protein increases σ(S) activity by favouring its association to E. Taking advantage of evolution of the σ(S) sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in σ(S) to the S. Typhimurium σ(S)-Crl binding interface. We solved the solution structure of S. Typhimurium Crl by NMR and used it for NMR binding assays with σ(S) and to generate in silico models of the σ(S)-Crl complex constrained by mutational analysis. The σ(S)-Crl models suggest that the identified arginine in σ(S) interacts with an aspartate of Crl that is required for σ(S) binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface. This study provides the basis for further structural investigation of the σ(S)-Crl complex.

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 σ 70 –RNAP and the λP R 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 σ 70 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. Time-resolved cryo-EM captured transient intermediates during E. coli RNAP promoter melting, revealing conformational changes affecting stepwise transcription bubble opening. Results inform how DNA sequence controls bacterial transcription initiation.

Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ 70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β’ lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ 1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ 1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp. Ribosomal RNA (rRNA) expression is regulated at the initiation stage of RNA synthesis. Here, the authors report cryo-EM structures of E. coli RNA polymerase and rRNA promoter complex with DksA/ppGpp on the way to open complex formation, identifying key steps in promoter recognition and opening.

After an RNA polymerase reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA and recommence RNA synthesis from the previous or a different promoter. Magnetic tweezers were used to monitor such secondary transcription and determine the effects of low forces assisting or opposing translocation, protein roadblocks, and transcription factors. Remarkably, up to 50% of Escherichia coli ( E. coli ) RNA polymerases diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 ( σ 70 ) likely remained associated with the DNA promoting sliding and enabling re-initiation from promoters in either orientation. However, deletions of the α -C-terminal domains severely limited the ability of RNAP to turn around between successive rounds of transcription. The addition of elongation factor NusG, which competes with σ 70 for binding to RNAP, limited additional rounds of transcription. Surprisingly, sliding RNA polymerases blocked by a DNA-bound lac repressor could slowly re-initiate transcription and were not affected by NusG, suggesting a σ -independent pathway. Low forces effectively biased promoter selection suggesting a prominent role for topological entanglements that affect RNA polymerase translocation. Tiny forces cause E. coli RNA polymerase to slide along DNA after transcription termination at up to 700 bp/s. In this paper, the authors use magnetic tweezers to show that sliding complexes containing complete alpha C-terminal domains repeat transcription from protein roadblocks sites and promoters in either orientation.

Multisubunit RNA polymerases (RNAPs) associate with initiation factors (σ in bacteria) to start transcription. The σ factors are responsible for recognizing and unwinding promoter DNA in all bacterial RNAPs. Here, we report two cryo-EM structures of cyanobacterial transcription initiation complexes at near-atomic resolutions. The structures show that cyanobacterial RNAP forms an “SI3-σ” arch interaction between domain 2 of σA (σ₂) and sequence insertion 3 (SI3) in the mobile catalytic domain Trigger Loop (TL). The “SI3-σ” arch facilitates transcription initiation from promoters of different classes through sealing the main cleft and thereby stabilizing the RNAP-promoter DNA open complex. Disruption of the “SI3-σ” arch disturbs cyanobacteria growth and stress response. Our study reports the structure of cyanobacterial RNAP and a unique mechanism for its transcription initiation. Our data suggest functional plasticity of SI3 and provide the foundation for further research into cyanobacterial and chloroplast transcription.

Bacterial RNA polymerase employs extra-cytoplasmic function (ECF) σ factors to regulate context-specific gene expression programs. Despite being the most abundant and divergent σ factor class, the structural basis of ECF σ factor-mediated transcription initiation remains unknown. Here, we determine a crystal structure of Mycobacterium tuberculosis ( Mtb ) RNAP holoenzyme comprising an RNAP core enzyme and the ECF σ factor σ H (σ H -RNAP) at 2.7 Å, and solve another crystal structure of a transcription initiation complex of Mtb σ H -RNAP (σ H -RPo) comprising promoter DNA and an RNA primer at 2.8 Å. The two structures together reveal the interactions between σ H and RNAP that are essential for σ H -RNAP holoenzyme assembly as well as the interactions between σ H -RNAP and promoter DNA responsible for stringent promoter recognition and for promoter unwinding. Our study establishes that ECF σ factors and primary σ factors employ distinct mechanisms for promoter recognition and for promoter unwinding. Extra-cytoplasmic function (ECF) σ factors allow bacteria to rapidly respond to stress conditions. Here the authors provide insights into the mechanism of bacterial stress-induced transcription by determining the crystal structures of the M. tuberculosis ECF σ factor bound RNA Polymerase (RNAP) holoenzyme and the ECF σ factor-RNAP transcription initiation complex.

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time. Bacterial transcription is terminated when RNA polymerases encounter terminator sequences. Using a single-molecule fluorescence assay, here the authors show that the release of transcript RNA precedes RNA polymerase dissociation and that the remaining RNA polymerase can reinitiate at nearby promoters.

Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)– promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σᴬ holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme.

Gene expression in Escherichia coli is controlled by well-established mechanisms that activate or repress transcription. Here, we identify CedA as an unconventional transcription factor specifically associated with the RNA polymerase (RNAP) σ 70 holoenzyme. Structural and biochemical analysis of CedA bound to RNAP reveal that it bridges distant domains of β and σ 70 subunits to stabilize an open-promoter complex. CedA does so without contacting DNA. We further show that cedA is strongly induced in response to amino acid starvation, oxidative stress and aminoglycosides. CedA provides a basal level of tolerance to these clinically relevant antibiotics, as well as to rifampicin and peroxide. Finally, we show that CedA modulates transcription of hundreds of bacterial genes, which explains its pleotropic effect on cell physiology and pathogenesis. Here, using proteomics, next-generation sequencing, biochemistry and cryo-EM, the authors delineate the role of CedA as an unconventional transcription factor in Escherichia coli, which protects from different stressors, including antibiotics, by regulating the transcriptional landscape.

In bacteria, RNA polymerase (RNAP) often pauses during the early stages of transcription initiation. The structural basis for these transient pauses remains unclear. Here, we present cryo-electron microscopy (cryo-EM) structures of the paused initiation complex (PIC) and initiation complex (IC) of Mycobacterium tuberculosis ( Mtb ), which include the RNAP core enzyme, the ECF σ factor σ E , transcription factor CarD, promoter DNA, and nascent RNA. Our structures with pre-melted scaffolds reveal an intermediate at the 6–7 nt stage compatible with a paused-like intermediate, associated with steric hindrance between the emerging RNA and the σ3.2 region. This clash triggers a swivel of the RNAP structural module and scrunching of the transcription bubble. We also observe positional rearrangement of the σ4 domain, suggesting a poised pre-escape state. In addition, complementary reconstructions with fully matched DNA scaffolds (N-IC and N-PIC) support the physiological relevance of the captured intermediates. Together, our results support the existence of a mechanistic checkpoint during transcription initiation and suggest an RNA-induced model how RNAP conformational dynamics regulate early transcription. Pausing during early transcription by bacterial RNA polymerase is not fully understood. Here, the authors determine the structures of the paused initiation complex in Mycobacterium tuberculosis, revealing how RNA and polymerase interactions create a transcription checkpoint.