Faculty Bibliography

Exonuclease (Exo) III was used to probe translocation states of RNA polymerase (RNAP) ternary elongation complexes (TECs). Escherichia coli RNAP stalls primarily in a post-translocation register that makes relatively slow excursions to a hyper-translocated state or to a pre-translocated state. Tagetitoxin (TGT) strongly inhibits hyper-translocation and inhibits backtracking, so, as indicated by Exo III mapping, TGT appears to stabilize both the pre- and probably a partially post-translocation state of RNAP. Because the pre-translocated to post-translocated transition is slow at many template positions, these studies appear inconsistent with a model in which RNAP makes frequent and rapid (i.e., millisecond phase) oscillations between pre- and post-translocation states. Nine nucleotides (9-nt) and 10-nt TECs, and TECs with longer nascent RNAs, have distinct translocation properties consistent with a 9-10 nt RNA/DNA hybrid. RNAP mutant proteins in the bridge helix and trigger loop are identified that inhibit or stimulate forward and backward translocation.

Transcription antiterminator RfaH alternates between closed (inactive) and open (activated) conformation. In this issue of Cell, Burmann et al. show that opening is accompanied by dramatic all-alpha to all-beta refolding of its C-terminal domain. Each of the folds has a distinct function: all-alpha-fold acts as a specificity determinant, directing RfaH to a small subset of operons, whereas the all-beta-fold recruits ribosome, thereby coupling RfaH-stimulated transcription to translation.

RNA polymerase is a ratchet machine that oscillates between productive and backtracked states at numerous DNA positions. Since its first description 15 years ago, backtracking-the reversible sliding of RNA polymerase along DNA and RNA-has been implicated in many critical processes in bacteria and eukaryotes, including the control of transcription elongation, pausing, termination, fidelity, and genome instability.

Most bacteria generate nitric oxide (NO) either aerobically by NO synthases or anaerobically from nitrite. Far from being a mere by-product of nitrate respiration, bacterial NO has diverse physiological roles. Many proteins undergo NO-mediated posttranslational modification (S-nitrosylation) in anaerobically grown Escherichia coli. The regulation of one such protein, OxyR, represents a redox signaling paradigm in which the same transcription factor controls different protective genes depending on its S-nitrosylation versus S-oxidation status. We discuss a structural model that may explain the remarkable stability and specificity of OxyR S-nitrosylation.

Riboswitches are RNA sensors that regulate gene expression upon binding specific metabolites or ions. Bacterial riboswitches control gene expression primarily by promoting intrinsic transcription termination or by inhibiting translation initiation. We now report a third general mechanism of riboswitch action: governing the ability of the RNA-dependent helicase Rho to terminate transcription. We establish that Rho promotes transcription termination in the Mg(2+)-sensing mgtA riboswitch from Salmonella enterica serovar Typhimurium and the flavin mononucleotide-sensing ribB riboswitch from Escherichia coli when the corresponding riboswitch ligands are present. The Rho-specific inhibitor bicyclomycin enabled transcription of the coding regions at these two loci in bacteria experiencing repressing concentrations of the riboswitch ligands in vivo. A mutation in the mgtA leader that favors the "high Mg(2+)" conformation of the riboswitch promoted Rho-dependent transcription termination in vivo and in vitro and enhanced the ability of the RNA to stimulate Rho's ATPase activity in vitro. These effects were overcome by mutations in a C-rich region of the mRNA that is alternately folded at high and low Mg(2+), suggesting a role for this region in regulating the activity of Rho. Our results reveal a potentially widespread mode of gene regulation whereby riboswitches dictate whether a protein effector can interact with the transcription machinery to prematurely terminate transcription.

In their commentary, Klyuyev and Vassylyev dispute a model of transcription inhibition by tagetitoxin (Tgt) proposed by us based on biochemical analysis and computational docking. We maintain that, although an alternative explanation can be provided for any single observation reported by us, taken together our results support a model in which Tgt acts by trapping the trigger loop (TL) in an inactive state (Artsimovitch et al.). ( 1) This model is consistent with all the data collected with a physiological target for the inhibitor, the transcription elongation complex (EC). The Tgt-binding pose in our model is indeed different from that observed in the structure of the Thermus thermophilus RNA polymerase (RNAP) holoenzyme in the absence of nucleic acids (Vassylyev et al. Nat Struct Mol Biol 2005; 12:1086). The latter can hardly be considered a dogma because (1) RNAP undergoes conformational changes in the course of the transcription cycle and during catalysis and (2) small molecules containing phosphates likely bind to several sites on RNAP, with the crystallographic site/pose not necessarily being the one most relevant mechanistically. Furthermore, the model proposed based on the Tgt/holoenzyme structure does not explain the inhibitor's effects on transcript elongation and RNAP translocation. These arguments necessitate further inquiry into the mechanism of inhibition by Tgt by techniques orthogonal to X-ray crystallography. In our opinion, elucidation of a molecular mechanism of any RNAP inhibitor and the follow-up design of more potent derivatives requires a combination of approaches, including genetics, biochemistry, biophysics, X-ray crystallography and computational analysis.

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 beta-synthase, cystathionine gamma-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

Tagetitoxin (Tgt) inhibits multisubunit chloroplast, bacterial, and some eukaryotic RNA polymerases (RNAPs). A crystallographic structure of Tgt bound to bacterial RNAP apoenzyme shows that Tgt binds near the active site but does not explain why Tgt acts only at certain sites. To understand the Tgt mechanism, we constructed a structural model of Tgt bound to the transcription elongation complex. In this model, Tgt interacts with the beta' subunit trigger loop (TL), stabilizing it in an inactive conformation. We show that (i) substitutions of the Arg residue of TL contacted by Tgt confer resistance to inhibitor; (ii) Tgt inhibits RNAP translocation, which requires TL movements; and (iii) paused complexes and a "slow" enzyme, in which the TL likely folds into an altered conformation, are resistant to Tgt. Our studies highlight the role of TL as a target through which accessory proteins and antibiotics can alter the elongation complex dynamics.