Identifying the bicyclomycin binding domain through biochemical analysis of antibiotic-resistant Rho proteins

Mutations M219K, S266A, and G337S in transcription termination factor Rho h ave been shown to confer resistance to the antibiotic bicyclomycin (BCM). A ll three His-tagged mutant Rho proteins exhibited similar K-m values for AT P; however, the V-max values at infinite ATP concentrations were one-fourth to one-third that for the His-tagged wild-type enzyme. BCM inhibition kine tics of poly(C)-dependent ATPase activity for the mutant proteins were non- competitive with respect to ATP (altering catalytic function but not ATP bi nding) and showed increased K-i values compared with His-tagged wildtype Rh o, M219K and G337S exhibited increased ratios of poly(U)/poly(C)-stimulated ATPase activity and lower apparent K-m values for ribo(C)(10) in the poly( dC) ribo(C)(10)ependent ATPase assay compared with His-tagged wild-type Rho . The S266A mutation did not show an increased poly(U)/poly(C) ATPase activ ity ratio and maintained approximately the same K-m for ribo(C)(10) in the poly(dC). ribo(C)(10)- dependent ATPase assay, The kinetic studies indicate d that M219K and G337S altered the secondary RNA binding domain in Rho wher eas the S266A mutation did not. Transcription termination assays for each m utant showed different patterns of Rho-terminated transcripts. Tyrosine sub stitution of Ser-266 led to BCM sensitivity intimating that an OH (hydroxyl ) moiety at this position is needed for BCM (binding) inhibition. Our resul ts suggest BCM binds to Rho at a site distinct from both the ATP and the pr imary RNA binding domains but close to the secondary RNA-binding (tracking) site and the ATP hydrolysis pocket.