Phase Variation of Flagella and Toxins in Clostridioides difficile is Mediated by Selective Rho-dependent Termination.

Clostridioides difficile is an intestinal pathogen that exhibits phase variation of flagella and toxins through inversion of the flagellar (flg) switch controlling flagellar and toxin gene expression. The transcription termination factor Rho preferentially inhibits swimming motility of bacteria with the 'flg-OFF' switch sequence. How C. difficile Rho mediates this selectivity was unknown. C. difficile Rho contains an N-terminal insertion domain (NID) which is found in a subset of Rho orthologues and confers diverse functions. Here we determined how Rho distinguishes between flg-ON and -OFF mRNAs and the roles of the NID and other domains of C. difficile Rho. Using in vitro ATPase assays, we determined that Rho specifically binds a region containing the left inverted repeat of the flg switch, but only of flg-OFF mRNA, indicating that differential termination is mediated by selective Rho binding. Using a suite of in vivo and in vitro assays in C. difficile, we determined that the NID is essential for Rho termination of flg-OFF mRNA, likely by influencing the ability to form stable hexamers, and the RNA binding domain is critical for flg-OFF specific termination. This work gives insight into the novel mechanism by which Rho interacts with flg mRNA to mediate phase variation of flagella and toxins in C. difficile and broadens our understanding of Rho-mediated termination in an organism with an AT-rich genome.

Step-by-Step Regulation of Productive and Abortive Transcription Initiation by Pyrophosphorolysis.

An understanding of the kinetics and mechanism of bacterial transcription initiation is needed to understand regulation of gene expression and advance fields from antibiotic discovery to promoter design. The step-by-step forward kinetics and mechanism of initiation and RNA-DNA hybrid growth, made irreversible by omitting pyrophosphate (PPi) byproduct, were determined recently for E. coli RNA polymerase (RNAP)-λPR promoter complexes. Strong position-dependences of overall rate constants (kcat/Km analogs) for each nucleotide-addition step were observed because of coupling of hybrid growth to disruption of promoter contacts, bubble closing, and RNAP escape. Here we investigate reversal of these steps (pyrophosphorolysis) at PPi concentrations ([PPi]) found in exponentially-growing cells. We quantify [PPi] effects on the amount and rate of synthesis of long (>10-mer, post-escape) and short (stalled, abortive) RNA to determine how PPi regulates initiation. Physiological [PPi] makes uridine incorporation and some other initiation steps significantly reversible. Physiological [PPi] reduces the fraction of RNAP-promoter complexes that productively initiate and the rate of RNA synthesis per productive complex, while increasing the fraction of complexes that abortively initiate, affecting abortive rates, and shifting the abortive-product distribution to shorter RNAs. Pyrophosphorolysis rates for some initiation complexes are orders of magnitude larger than for removal of the same nucleotide from elongation complexes because of the strong bias toward the pre-translocated state in initiation, and exhibit even stronger dependences on nucleotide identity (pyrimidine ≫ purine). Because cytoplasmic [PPi] is much higher in exponential-phase than stationary-phase cells, these [PPi] effects on initiation rates and amounts of RNA synthesis must be physiologically-relevant.

Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise.

Transcription initiation is highly regulated by promoter sequence, transcription factors, and ligands. All known transcription inhibitors, an important class of antibiotics, act in initiation. To understand regulation and inhibition, the biophysical mechanisms of formation and stabilization of the "open" promoter complex (OC), of synthesis of a short RNA-DNA hybrid upon nucleotide addition, and of escape of RNA polymerase (RNAP) from the promoter must be understood. We previously found that RNAP forms three different OC with λPR promoter DNA. The 37 °C RNAP-λPR OC (RPO) is very stable. At lower temperatures, RPO is less stable and in equilibrium with an intermediate OC (I3). Here, we report step-by-step rapid quench-flow kinetic data for initiation and growth of the RNA-DNA hybrid at 25 and 37 °C that yield rate constants for each step of productive nucleotide addition. Analyzed together, with previously published data at 19 °C, our results reveal that I3 and not RPO is the productive initiation complex at all temperatures. From the strong variations of rate constants and activation energies and entropies for individual steps of hybrid extension, we deduce that contacts of RNAP with the bubble strands are disrupted stepwise as the hybrid grows and translocates. Stepwise disruption of RNAP-strand contacts is accompanied by stepwise bubble collapse, base stacking, and duplex formation, as the hybrid extends to a 9-mer prior to disruption of upstream DNA-RNAP contacts and escape of RNAP from the promoter.

Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by Escherichia coli RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex.

FRET (fluorescence resonance energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of λPR promoter DNA on Escherichia coli RNA polymerase (RNAP) in closed and open complexes (CC and OC, respectively). Here we determine the kinetics and mechanism of DNA bending and wrapping by FRET and of formation of RNAP contacts with -100 and +14 DNA by single-dye protein-induced fluorescence enhancement (PIFE). FRET and PIFE kinetics exhibit two phases: rapidly reversible steps forming a CC ensemble ({CC}) of four intermediates [initial (RPC), early (I1E), mid (I1M), and late (I1L)], followed by conversion of {CC} to OC via I1L. FRET and PIFE are first observed for I1E, not RPc. FRET and PIFE together reveal large-scale bending and wrapping of upstream and downstream DNA as RPC advances to I1E, decreasing the Cy3-Cy5 distance to ∼75 Å and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper ß'-clamp while downstream DNA contacts the top of the ß-pincer in I1E. Converting I1E to I1M (∼1 s time scale) reduces FRET efficiency with little change in -100 or +14 PIFE, interpreted as clamp opening that moves far-upstream DNA (on ß') away from downstream DNA (on ß) to increase the Cy3-Cy5 distance by ∼14 Å. FRET increases greatly in converting I1M to I1L, indicating bending of downstream duplex DNA into the clamp and clamp closing to reduce the Cy3-Cy5 distance by ∼21 Å. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I1L is converted to the initial unstable OC (I2). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream binding, DNA-bending transcription activators are discussed.