Isolation and sequencing of the rho gene from Streptomyces lividans ZX7 and characterization of the RNA-dependent NTPase activity of the overexpressed protein

C J Ingham, Irene Hunter, Margaret Caroline MacHin Smith

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)21803-21807
Number of pages5
JournalThe Journal of Biological Chemistry
Volume271
Issue number36
Publication statusPublished - 6 Sep 1996

Keywords

  • TERMINATION FACTOR-RHO
  • TRANSCRIPTION TERMINATION
  • ESCHERICHIA-COLI
  • ATPASE ACTIVITY
  • IDENTIFICATION
  • BICYCLOMYCIN
  • EFFICIENCY
  • HELICASE
  • BACTERIA
  • DOMAIN

Cite this