Analysis of E. coli rho factor: mutations affecting secondary-site interactions.
To define and differentiate primary and secondary RNA binding sites within the linear sequence of the rho protein, we investigated two mutant alleles, rho-115 and rhosuA1. They were first identified as defective in transcription termination in vivo, and later demonstrated to be defective in their interactions with RNA at the primary and secondary sites, respectively. Sequencing of rhosuA1 revealed a single lysine to glutamic acid residue change at position 352 (KE352), while rho-115 carries two mutations, glycine99 to valine (GV99) and a proline235 to histidine (PH235). Proteins carrying single mutations at each of these three positions were purified and their characteristics compared to the wild-type protein. We found both KE352 and GV99 to be defective in secondary-site RNA activation, with Km values for r(C)10 of 100 microM and approximately 650 microM, respectively, compared to the wild-type value of 4 microM. These observed secondary-site defects correlated with decreased helicase and ATPase activities, as well as a loss of transcription termination activity in vitro. By contrast, PH235 was very efficient at interacting with r(C)10 at the secondary site, with a measured Km of 0.5 microM, and displayed the characteristics of a hyperactive rho, as judged by its ATPase, helicase and termination capabilities. Our results show that mutations at three very different locations in the polypeptide can affect secondary-site activation by RNA, and that these interactions play a pivotal role in ATP hydrolysis, helicase activity and transcription termination.
Pereira
S
S
Department of Biochemistry, University of Rochester Medical Center, NY 14642, USA.
Platt
T
T
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001665:Binding Sites; D004265:DNA Helicases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D009154:Mutation; D011066:Poly C / Q000378:metabolism; D012364:RNA, Transfer, Trp / Q000235:genetics / Q000378:metabolism; D012234:Rho Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic
D012364:RNA, Transfer, Trp; D012234:Rho Factor; D011066:Poly C; D000251:Adenosine Triphosphatases; D004265:DNA Helicases