Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs

Mol Cell. 2021 May 20;81(10):2201-2215.e9. doi: 10.1016/j.molcel.2021.04.027.


The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and demonstrate that some RNA polymerase mutants both decrease average cell length and prevent killing by cell-wall targeting antibiotics. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships in vivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.

Keywords: A22; FtsZ; RNAP; SI2; chemical genetics; lineage-specific sequence insertion; mecillinam; stringent response; transcription.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amdinocillin / pharmacology
  • Bacterial Proteins / metabolism
  • Cell Death / drug effects
  • Chromosomes, Bacterial / genetics
  • Cytoprotection / drug effects
  • Cytoskeletal Proteins / metabolism
  • DNA-Directed RNA Polymerases / chemistry*
  • DNA-Directed RNA Polymerases / genetics*
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial / drug effects
  • Mutagenesis, Insertional / genetics
  • Mutation / genetics*
  • Peptides / metabolism
  • Phenotype
  • Structure-Activity Relationship
  • Transcription, Genetic
  • Uridine Diphosphate Glucose / metabolism


  • Bacterial Proteins
  • Cytoskeletal Proteins
  • FtsZ protein, Bacteria
  • Peptides
  • DNA-Directed RNA Polymerases
  • Amdinocillin
  • Uridine Diphosphate Glucose