Faculty Bibliography

Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering¹ to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.

Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Traditional models of transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryoelectron microscopy (cryo-EM) structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific "pre-termination complex" (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC. RNA exiting RNAP interacts with NusA before entering the central channel of Rho from the distal C-terminal side of the ring. We map the principal interactions in the PTC and demonstrate their critical role in termination. Our results support a mechanism in which the formation of a persistent PTC is a prerequisite for termination.

In search for RNA signals that modulate transcription via direct interaction with RNA polymerase (RNAP), we deep sequenced an E. coli genomic library enriched for RNAP-binding RNAs. Many natural RNAP-binding aptamers, termed RAPs, were mapped to the genome. Over 60% of E. coli genes carry RAPs in their mRNA. Combining in vitro and in vivo approaches, we characterized a subset of inhibitory RAPs (iRAPs) that promote Rho-dependent transcription termination. A representative iRAP within the coding region of the essential gene, nadD, greatly reduces its transcriptional output in stationary phase and under oxidative stress, demonstrating that iRAPs control gene expression in response to changing environment. The mechanism of iRAPs involves active uncoupling of transcription and translation, making nascent RNA accessible to Rho. iRAPs encoded in the antisense strand also promote gene expression by reducing transcriptional interference. In essence, our work uncovers a broad class of cis-acting RNA signals that globally control bacterial transcription.

Transcription-coupled repair (TCR) serves an important role in preserving genome integrity and maintaining fidelity of replication. Coupling transcription to DNA repair requires a coordinated action of several factors, including transcribing RNA polymerase and various transcription modulators and repair proteins. To study TCR in molecular detail, it is important to employ defined protein complexes in vitro and defined genetic backgrounds in vivo. In this chapter, we present methods to interrogate various aspects of TCR at different stages of repair. We describe promoter-initiated and nucleic acid scaffold-initiated transcription as valid approaches to recapitulate various stages of TCR, and discuss their strengths and weaknesses. We also outline an approach to study TCR in its cellular context using Escherichia coli as a model system.

Bacterial small RNAs (sRNAs) have been implicated in various aspects of post-transcriptional gene regulation. Here, we demonstrate that sRNAs also act at the level of transcription termination. We use the rpoS gene, which encodes a general stress sigma factor sigma(S), as a model system, and show that sRNAs DsrA, ArcZ, and RprA bind the rpoS 5'UTR to suppress premature Rho-dependent transcription termination, both in vitro and in vivo. sRNA-mediated antitermination markedly stimulates transcription of rpoS during the transition to the stationary phase of growth, thereby facilitating a rapid adjustment of bacteria to global metabolic changes. Next generation RNA sequencing and bioinformatic analysis indicate that Rho functions as a global "attenuator" of transcription, acting at the 5'UTR of hundreds of bacterial genes, and that its suppression by sRNAs is a widespread mode of bacterial gene regulation.

The small molecule alarmone (p)ppGpp mediates bacterial adaptation to nutrient deprivation by altering the initiation properties of RNA polymerase (RNAP). ppGpp is generated in Escherichia coli by two related enzymes, RelA and SpoT. We show that ppGpp is robustly, but transiently, induced in response to DNA damage and is required for efficient nucleotide excision DNA repair (NER). This explains why relA-spoT-deficient cells are sensitive to diverse genotoxic agents and ultraviolet radiation, whereas ppGpp induction renders them more resistant to such challenges. The mechanism of DNA protection by ppGpp involves promotion of UvrD-mediated RNAP backtracking. By rendering RNAP backtracking-prone, ppGpp couples transcription to DNA repair and prompts transitions between repair and recovery states.

Precise transcription by cellular RNA polymerase requires efficient removal of onn-cognate nucleotide residues occasionally incorporated. Mis-incorporation causes transcription elongation complex (TEC) to backtrack, releasing single strand 3 prime RNA segment bearing non-cognate residue, which is hydrolyzed by the active center that carries two Mg2+ ions. However, in most X-ray structures only one Mg2+ is present. This Mg2+ is tightly bound to the active center aspartates, creating inactive stable state. The first residue of single strand RNA segment in backtracked TEC strongly promotes transcript hydrolytic cleavage by establishing a network of interactions that force a shift of stably bound Mg2+ to release some of its aspartate coordination valences for binding to the second Mg2+ enabling catalysis. Such rearrangement that we call active center tuning (ACT) occurs when all recognition contacts of active center-bound RNA segment are established and verified by tolerance to stress. Transcription factor Gre builds on ACT mechanism in the same reaction by increasing the retention of the second Mg2+ and by activating the attacking water, causing 3000-4000 fold reaction acceleration and strongly reinforcing proofreading. Unified mechanism for RNA synthesis and degradation by RNAP predicts that ACT also executes NTP selection thereby contributing to high transcription fidelity.

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.

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.