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Volume 271, Number 41, Issue of October 11, 1996 pp. 25369-25374
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, April 5, 1996, and in revised form, June 24, 1996)

Attila Magyar Dagger , Xiangdong Zhang § , Harold Kohn § and William R. Widger Dagger

From the Dagger  Department of Biochemical and Biophysical Sciences, University of Houston, Houston, Texas 77204-5934 and § Department of Chemistry, University of Houston, Houston, Texas 77204-5641

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.




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