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 M...
Gespeichert in:
Veröffentlicht in: | The Journal of biological chemistry 1996-09, Vol.271 (36), p.21803-21807 |
---|---|
Hauptverfasser: | , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
Zusammenfassung: | 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. |
---|---|
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.271.36.21803 |