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Volume 271, Number 36, Issue of September 6, 1996 pp. 21803-21807
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Isolation and Sequencing of the rho Gene from Streptomyces lividans ZX7 and Characterization of the RNA-dependent NTPase Activity of the Overexpressed Protein

(Received for publication, March 6, 1996, and in revised form, May 22, 1996)

Colin J. Ingham , Iain S. Hunter and Margaret C. M. Smith

From the  Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12, United Kingdom and the  Department of Genetics, Queens Medical Centre, Nottingham University, Nottingham, NG7 2UH United Kingdom

The gene for transcription termination factor Rho was isolated from Streptomyces lividans ZX7. It encoded a 77-kDa polypeptide (Rho 77) with considerable homology to known Rho factors. An atypical hydrophilic region of 228 residues was found within the N-terminal RNA-binding domain. Only Rho from Micrococcus luteus and Mycobacterium leprae (closely related GC-rich Gram-positive bacteria) had an analogous sequence. Rho 77 was overexpressed in Escherichia coli and purified using an N-terminal hexahistidine-tag. Rho 77 displayed a broad RNA-dependent ATPase activity, with poly(C) RNA being no more than 4-fold more effective than poly(A). This contrasts with the ATPase activity of Rho from E. coli which is stimulated primarily by poly(C) RNA. Rho 77 was a general RNA-dependent NTPase, apparent K_m values for NTPs were: GTP 0.13 mM, ATP 0.17 mM, UTP 1.1 mM, and CTP >2 mM. Rho 77 poly(C)-dependent ATPase activity was inhibited by heparin, unlike the E. coli Rho. The antibiotic bicyclomycin inhibited the in vitro RNA-dependent ATPase activity of Rho 77, did not inhibit growth of streptomycetes but delayed the development of aerial mycelia. N-terminal deletion analysis to express a truncated form of Rho (Rho 72, 72 kDa) indicated that the first 42 residues of Rho 77 were not essential for RNA-dependent NTPase activity and were not the targets of inhibition by heparin or bicyclomycin.

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