Simple enzymatic assays for the in vitro motor activity of transcription termination factor Rho from Escherichia coli.
The transcription termination factor Rho from Escherichia coli is a ring-shaped homo-hexameric protein that preferentially interacts with naked cytosine-rich Rut (Rho utilization) regions of nascent RNA transcripts. Once bound to the RNA chain, Rho uses ATP as an energy source to produce mechanical work and disruptive forces that ultimately lead to the dissociation of the ternary transcription complex. Although transcription termination assays have been useful to study Rho activity in various experimental contexts, they do not report directly on Rho mechanisms and kinetics. Here, we describe complementary ATP-dependent RNA-DNA helicase and streptavidin displacement assays that can be used to monitor in vitro Rho's motor activity in a more direct and quantitative manner.
Boudvillain
Marc
M
Centre de Biophysique Moleculaire (UPR4301), CNRS, Orleans cedex 2, Orleans, France.
Walmacq
Céline
C
Schwartz
Annie
A
Jacquinot
Frédérique
F
D001681:Biological Assay / Q000379:methods*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012234:Rho Factor / Q000235:genetics / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012234:Rho Factor; D019809:Streptavidin; D004265:DNA Helicases; D020365:RNA Helicases