Analyzing ATP utilization by DEAD-Box RNA helicases using kinetic and equilibrium methods.
DEAD-box proteins (DBPs) couple ATP utilization to conformational rearrangement of RNA. In this chapter, we outline a combination of equilibrium and kinetic methods that have been developed and applied to the analysis of ATP utilization and linked RNA remodeling by DBPs, specifically Escherichia coli DbpA and Saccharomyces cerevisiae Mss116. Several important considerations are covered, including solution conditions, DBP assembly/aggregation, and RNA substrate properties. We discuss practical experimental methods for determination of DBP-RNA-nucleotide binding affinities and stoichiometries, steady-state ATPase activity, ATP binding, hydrolysis and product release rate constants, and RNA unwinding. We present general methods to integrate and analyze this combination of experimental data to identify the preferred kinetic pathway of ATP utilization and linked dsRNA unwinding.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
Michael J
MJ
Bradley
Enrique M
EM
De La Cruz
D000255:Adenosine Triphosphate / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D007700:Kinetics; D020871:RNA Stability; D013696:Temperature; D013816:Thermodynamics
D000255:Adenosine Triphosphate; D053487:DEAD-box RNA Helicases