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 E. coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations dramatically 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 functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature. ChIP-seq data from rifampicin hypersensitive mutants after treatment and recovery from rifampicin.