ANALYSIS OF ESCHERICHIA-COLI RHO-FACTOR - MUTATIONS AFFECTING SECONDARY-SITE INTERACTIONS

To define and differentiate primary and secondary RNA binding sites wi thin the linear sequence of the rho protein, we investigated two mutan t alleles, rho-115 and rhosuA1. They were first identified as defectiv e in transcription termination in vivo, and later demonstrated to be d efective in their interactions with RNA at the primary and secondary s ites, respectively. Sequencing of rhosuA1 revealed a single lysine to glutamic acid residue change at position 352 (KE352), while rho-115 ca rries two mutations, glycine99 to valine (GV99) and a proline235 to hi stidine (PH235). Proteins carrying single mutations at each of these t hree positions were purified and their characteristics compared to the wild-type protein. We found both KE352 and GV99 to be defective in se condary-site RNA activation, with K-m values for r(C)(10) of 100 mu M and similar to 650 mu M, respectively, compared to the wild-type value of 4 mu M. These observed secondary-site defects correlated with decr eased helicase and ATPase activities, as well as a loss of transcripti on termination activity in vitro. By contrast, PH235 was very efficien t at interacting with r(C)io at the secondary site, with a measured K- m of 0.5 mu M, and displayed the characteristics of a hyperactive rho, as judged by its ATPase, helicase and termination capabilities. Our r esults show that mutations at three very different locations in the po lypeptide can affect secondary-site activation by RNA, and that these interactions play a pivotal role in ATP hydrolysis, helicase activity and transcription termination. (C) 1995 Academic Press Limited