Isolation and sequencing of the rho gene from Streptomyces lividans ZX7 and characterization of the RNA-dependent NTPase activity of the overexpressed protein.
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 Km 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.
Ingham
C J
CJ
Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12, United Kingdom.
Hunter
I S
IS
Smith
M C
MC
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis; D001483:Base Sequence; D002874:Chromosome Mapping; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D016982:Micrococcus luteus; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009166:Mycobacterium leprae; D012234:Rho Factor / Q000235:genetics*; D012293:Rifampin / Q000494:pharmacology; D016930:Saccharopolyspora; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D015010:Yersinia pestis
D012234:Rho Factor; D006493:Heparin; D000251:Adenosine Triphosphatases; C031715:rho-ATPase; D012293:Rifampin