A mutation in the ATP binding domain of rho alters its RNA binding properties and uncouples ATP hydrolysis from helicase activity.
The Escherichia coli mutant rho201 was originally isolated in a genetic screen for defects in rho-dependent termination. Cloning and sequencing of this gene reveals a single phenylalanine to cysteine mutation at residue 232 in the ATP binding domain of the protein. This mutation significantly alters its RNA binding properties so that it binds trp t', RNA 100-fold weaker than the wild type protein, with a Kd of approximately 1.3 nM. Rho201 binds nonspecific RNA only 3-4-fold less tightly than it binds trp t', while the wild type differential for these same RNAs is 10-20-fold. Curiously, rho201 displays increased secondary site RNA activation, with a Km for ribo(C)10 of 0.6 microM, compared to the wild type value of 3-4 microM. Although rho201 and the wild type protein hydrolyze ATP similarly with poly(C), or trp t' RNA, as cofactors, rho201 has a higher ATPase activity when activated by nonspecific RNA. Physically, rho201 displays an abnormal conformation detectable by mild trypsin digestion. Despite effective ATP hydrolysis, the rho201 mutant is a poor RNA:DNA helicase and terminates inefficiently on trp t'. The single F232C mutation thus appears to uncouple the protein's ATPase activity from its helicase function, so rho can no longer harness available energy for use in subsequent reactions.
Pereira
S
S
Department of Biochemistry, University of Rochester Medical Center, New York 14642, USA.
Platt
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012234:Rho Factor / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D011994:Recombinant Proteins; D012234:Rho Factor; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases; D020365:RNA Helicases