THE ANTIBIOTIC BICYCLOMYCIN AFFECTS THE SECONDARY RNA-BINDING SITE OFESCHERICHIA-COLI TRANSCRIPTION TERMINATION FACTOR-RHO

The interaction of Rho and the antibiotic bicyclomycin was probed usin g in vitro transcription termination reactions, poly(C) binding assays , limited tryptic digestions, and the bicyclomycin inhibition kinetics of ATPase activity in the presence of poly(dC) and ribo(C)(10). The a pproximate I-50 value for the bicyclomycin inhibition of transcription termination at Rho-dependent sites within a modified trp operon templ ate was 5 mu M. At antibiotic concentrations near the I-50 value, bicy clomycin inhibition of Rho-dependent transcripts was accompanied by th e appearance of a new set of transcripts whose size was midway between the Rho-dependent transcripts and the readthrough transcripts, Bicycl omycin did not inhibit poly(C) binding to Rho, In the presence of poly (dC), bicyclomycin showed a reversible mixed inhibition of the ribo(C) (10)-stimulated ATPase activity, The extrapolated K-i for bicyclomycin was 2.8 mu M without ribo(C)(10) and increased to 26 mu M in the pres ence of ribo(C)(10). Correspondingly, the K-m(app) for ribo(C)(10) wit hout bicyclomycin was 0.8 mu M and with bicyclomycin was 5 mu M at inf inite inhibitor concentration, The data suggested that the antibiotic binds to Rho, influencing the secondary RNA binding (tracking) site on Rho and slows the tracking of Rho toward the bound RNA polymerase.