Riboswitches were discovered in 2002 in bacteria as RNA-based intracellular sensors of vitamin derivatives. During the last decade, naturally occurring RNA sensor elements have been found to bind a range of small metabolites and ions and to exert regulatory control of transcription, translation, splicing, and RNA stability. Extensive biochemical, structural, and genetic studies have established the basic principles underpinning riboswitch function in all three kingdoms of life with implications for developing antibiotics, designing new molecular sensors, and integrating riboswitches into synthetic circuits.

Many prokaryotic species generate hydrogen sulfide (H(2)S) in their natural environments. However, the biochemistry and physiological role of this gas in nonsulfur bacteria remain largely unknown. Here we demonstrate that inactivation of putative cystathionine β-synthase, cystathionine γ-lyase, or 3-mercaptopyruvate sulfurtransferase in Bacillus anthracis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli suppresses H(2)S production, rendering these pathogens highly sensitive to a multitude of antibiotics. Exogenous H(2)S suppresses this effect. Moreover, in bacteria that normally produce H(2)S and nitric oxide, these two gases act synergistically to sustain growth. The mechanism of gas-mediated antibiotic resistance relies on mitigation of oxidative stress imposed by antibiotics.

During transcription of protein-coding genes, bacterial RNA polymerase (RNAP) is closely followed by a ribosome that is engaged in translation of the newly synthesized transcript. Here we show that as a result of translation-transcription coupling the overall elongation rate of transcription is tightly controlled by translation. Acceleration and deceleration of a ribosome results in corresponding changes in the speed of RNAP. Consistently, we found an inverse correlation between the number of rare codons in a gene, which delay ribosome progression, and the rate of transcription. We further show that the stimulating effect of a ribosome on RNAP is achieved by preventing RNAP from adopting non-productive states. The moving ribosome inhibits spontaneous backtracking of RNAP, thereby enhancing its pace and also facilitating read-through of roadblocks in vivo. Such a cooperative mechanism ensures the two gene expression machineries match precisely each other rates, so that the transcriptional yield is always adjusted to translational needs at different genes and under various growth conditions.

RNA chain elongation is a highly processive and accurate process that is finely regulated by numerous intrinsic and extrinsic signals. Here we describe a general mechanism that governs RNA polymerase (RNAP) movement and response to regulatory inputs such as pauses, terminators, and elongation factors. We show that E.coli RNAP moves by a complex Brownian ratchet mechanism, which acts prior to phosphodiester bond formation. The incoming substrate and the flexible F bridge domain of the catalytic center serve as two separate ratchet devices that function in concert to drive forward translocation. The adjacent G loop domain controls F bridge motion, thus keeping the proper balance between productive and inactive states of the elongation complex. This balance is critical for cell viability since it determines the rate, processivity, and fidelity of transcription.

An 8-9 bp RNA-DNA hybrid in the transcription elongation complex is essential for keeping the RNA 3' terminus engaged with the active site of E. coli RNA polymerase (RNAP). Destabilization of the hybrid leads to detachment of the transcript terminus, RNAP backtracking, and shifting of the hybrid upstream. Eventually, the exposed 3' segment of RNA can be removed through transcript cleavage. At certain sites, cycles of unwinding-rewinding of the hybrid are coupled to reverse-forward sliding of the transcription elongation complex. This explains apparent discontinuous elongation, which was previously interpreted as contraction and expansion of an RNAP molecule (inch-worming). Thus, the 3'-proximal RNA-DNA hybrid plays the dual role of keeping the active site in register with the template and sensing the helix-destabilizing mismatches in RNA, launching correction through backtracking and cleavage.

SUMMARY Frequent co-directional collisions between the replisome and RNA polymerase (RNAP) are inevitable because the rate of replication is much faster than that of transcription. Here we show that the outcome of such collisions depends on the productive state of transcription elongation complexes (ECs). Co-directional collisions with backtracked (arrested) ECs lead to DNA double strand breaks (DSBs), whereas head-on collisions do not. A mechanistic model is proposed to explain backtracking-mediated DSBs. We further show that bacteria employ various strategies to evade replisome collisions with backtracked RNAP, the most general of which is translation that prevents RNAP backtracking. If translation is abrogated, DSBs are suppressed by elongation factors that either prevent backtracking or reactivate backtracked ECs. Finally, termination factors also contribute to genomic stability by removing arrested ECs. Our results establish RNAP backtracking as the intrinsic hazard to chromosomal integrity and implicate active ribosomes and other anti-backtracking mechanisms in genome maintenance.

Transcription of the bacterial genome by the RNA polymerase must terminate at specific points. Transcription can be terminated by Rho factor, an essential protein in enterobacteria. We used the antibiotic bicyclomycin, which inhibits Rho, to assess its role on a genome-wide scale. Rho is revealed as a global regulator of gene expression that matches Escherichia coli transcription to translational needs. We also found that genes in E. coli that are most repressed by Rho are prophages and other horizontally acquired portions of the genome. Elimination of these foreign DNA elements increases resistance to bicyclomycin. Although rho remains essential, such reduced-genome bacteria no longer require Rho cofactors NusA and NusG. Deletion of the cryptic rac prophage in wild-type E. coli increases bicyclomycin resistance and permits deletion of nusG. Thus, Rho termination, supported by NusA and NusG, is required to suppress the toxic activity of foreign genes.

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