High-resolution landscape of an antibiotic binding site.

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.

RNA polymerase drives ribonucleotide excision DNA repair in E. coli.

Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside monophosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull-downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy structures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show specific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states. The weakening of RNAP-RNaseHII interactions compromises RER in vivo. The structure-functional data support a model where RNaseHII scans DNA in one dimension in search for rNMPs while "riding" the RNAP. We further demonstrate that TC-RER accounts for a significant fraction of repair events, thereby establishing RNAP as a surveillance "vehicle" for detecting the most frequently occurring replication errors.

Control of transcription elongation and DNA repair by alarmone ppGpp.

Second messenger (p)ppGpp (collectively guanosine tetraphosphate and guanosine pentaphosphate) mediates bacterial adaptation to nutritional stress by modulating transcription initiation. More recently, ppGpp has been implicated in coupling transcription and DNA repair; however, the mechanism of ppGpp engagement remained elusive. Here we present structural, biochemical and genetic evidence that ppGpp controls Escherichia coli RNA polymerase (RNAP) during elongation via a specific site that is nonfunctional during initiation. Structure-guided mutagenesis renders the elongation (but not initiation) complex unresponsive to ppGpp and increases bacterial sensitivity to genotoxic agents and ultraviolet radiation. Thus, ppGpp binds RNAP at sites with distinct functions in initiation and elongation, with the latter being important for promoting DNA repair. Our data provide insights on the molecular mechanism of ppGpp-mediated adaptation during stress, and further highlight the intricate relationships between genome stability, stress responses and transcription.

Crucial role and mechanism of transcription-coupled DNA repair in bacteria.

Transcription-coupled DNA repair (TCR) is presumed to be a minor sub-pathway of nucleotide excision repair (NER) in bacteria. Global genomic repair is thought to perform the bulk of repair independently of transcription. TCR is also believed to be mediated exclusively by Mfd-a DNA translocase of a marginal NER phenotype1-3. Here we combined in cellulo cross-linking mass spectrometry with structural, biochemical and genetic approaches to map the interactions within the TCR complex (TCRC) and to determine the actual sequence of events that leads to NER in vivo. We show that RNA polymerase (RNAP) serves as the primary sensor of DNA damage and acts as a platform for the recruitment of NER enzymes. UvrA and UvrD associate with RNAP continuously, forming a surveillance pre-TCRC. In response to DNA damage, pre-TCRC recruits a second UvrD monomer to form a helicase-competent UvrD dimer that promotes backtracking of the TCRC. The weakening of UvrD-RNAP interactions renders cells sensitive to genotoxic stress. TCRC then recruits a second UvrA molecule and UvrB to initiate the repair process. Contrary to the conventional view, we show that TCR accounts for the vast majority of chromosomal repair events; that is, TCR thoroughly dominates over global genomic repair. We also show that TCR is largely independent of Mfd. We propose that Mfd has an indirect role in this process: it participates in removing obstructive RNAPs in front of TCRCs and also in recovering TCRCs from backtracking after repair has been completed.