Effects of reaction conditions on RNA secondary structure and on the helicase activity of Escherichia coli transcription termination factor Rho.
The ATPase and helicase activities of the Escherichia coli transcription termination protein rho have been studied under a variety of reaction conditions that alter its transcription termination activity. These conditions include KCl, KOAc, or KGlu concentrations from 50 to 150 mM and Mg(OAc)2 concentrations from 1 to 5 mM (in the presence of 1 mM ATP). In higher KCl or higher Mg(OAc)2 concentrations we found that the translocation of rho hexamers along RNA was slower and less processive than the same process measured at 50 mM monovalent salt concentrations and 1 mM Mg(OAc)2. The ATPase activity of rho was also decreased under reaction conditions that slowed translocation. RNA melting experiments showed that the decreased ATPase activity of rho and the slower helicase activity at increased KCl or Mg(OAc)2 concentrations are accompanied by a concomitant increase in the secondary structure of the RNA portion of the helicase substate. In contrast, the ATPase activity of rho in the presence of poly(rC), a synthetic RNA that does not form salt-concentration-dependent secondary structure, was shown to be the same in each of the three monovalent salts. Thus, the salts do not directly affect the structure or conformation of the rho protein or the binding of rho to single-stranded RNA. However, the translocation of rho along RNA was more processive in 150 mM KOAc or KGlu than in 150 mM KCl, while the RNA secondary structure was the same in all three monovalent salts. Therefore, the monovalent salt present in the reaction may directly affect rho-RNA interactions when the RNA substrate can form secondary structure. Helicase experiments with an RNA molecule that does not contain a rho loading-site showed that rho translocates less processively along this potential helicase substrate. These results suggest that the helicase activity of rho may be significantly regulated by RNA secondary structure. In addition, one of the mechanisms to concentrate the activity of rho on transcripts containing unstructured rho loading sites may be that rho translocation along such molecules is more processive than it is along more structured RNA molecules in the cell.
Walstrom
K M
KM
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403, USA.
Dozono
J M
JM
von Hippel
P H
PH
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D008274:Magnesium; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011189:Potassium Chloride; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D012234:Rho Factor / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012234:Rho Factor; D011189:Potassium Chloride; D012316:RNA Nucleotidyltransferases; D020365:RNA Helicases; D008274:Magnesium