Using multiplex genome engineering to overcome the constraint of positive selection, this study generates and characterizes a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of *Escherichia coli* RNA polymerase (RNAP). The research identifies an alpha helix where mutations significantly enhance or disrupt rifampicin binding, leading to the discovery of mutations that convert rifampicin from bacteriostatic to bactericidal by inducing lethal DNA breaks. Additional mutations are found to increase RNAP speed, impacting nucleotide depletion, antibiotic sensitivity, and cold growth advantage. Finally, the study maps natural *rpoB* sequence diversity, revealing frequent occurrence of functional rifampicin binding site mutations in nature.

Kevin B. Yang, Maria Cameranesi, Manjunath Gowder, Criseyda Martinez, Yosef Shamovsky, Vitaliy Epshtein, Zhitai Hao, Thao Nguyen, Eric Nirenstein, Ilya Shamovsky, Aviram Rasouly, Evgeny Nudler