The Antibiotic Bicyclomycin Affects the Secondary RNA Binding Site of Escherichia coli Transcription Termination Factor Rho

• Published in: The Journal of biological chemistry 1996-10, Vol.271 (41), p.25369-25374
• Main Authors: Magyar, Attila, Zhang, Xiangdong, Kohn, Harold, Widger, William R.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Anti-Bacterial Agents - pharmacology; Binding Sites; Bridged Bicyclo Compounds, Heterocyclic - pharmacology; DNA-Directed RNA Polymerases - metabolism; Escherichia coli; Escherichia coli - metabolism; Molecular Structure; Operon; Peptide Fragments - metabolism; Poly C - metabolism; Rho Factor - chemistry; Rho Factor - metabolism; RNA - metabolism; Transcription, Genetic - drug effects
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.41.25369

The interaction of Rho and the antibiotic bicyclomycin was probed using 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 approximate I50 value for the bicyclomycin inhibition of transcription termination at Rho-dependent sites within a modified trp operon template was 5 μM. At antibiotic concentrations near the I50 value, bicyclomycin inhibition of Rho-dependent transcripts was accompanied by the appearance of a new set of transcripts whose size was midway between the Rho-dependent transcripts and the readthrough transcripts. Bicyclomycin 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 Ki for bicyclomycin was 2.8 μM without ribo(C)10 and increased to 26 μM in the presence of ribo(C)10. Correspondingly, the Km(app) for ribo(C)10 without bicyclomycin was 0.8 μM and with bicyclomycin was 5 μM at infinite 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.