ANALYZING ATP UTILIZATION BY DEAD-BOX RNA HELICASES USING KINETIC AND EQUILIBRIUM METHODS

被引：12

作者：

Bradley, Michael J. ^{[1
]}

De La Cruz, Enrique M. ^{[1
]}

机构：

[1] Yale Univ, Dept Mol Biophys & Biochem, New Haven, CT 06520 USA

来源：

RNA HELICASES | 2012年 / 511卷

关键词：

23S RIBOSOMAL-RNA; NUCLEOTIDE-BINDING SITES; ESCHERICHIA-COLI; DNA HELICASES; ADENOSINE-TRIPHOSPHATASE; TRANSLATION INITIATION; FLUORESCENT ANALOGS; PHALLOIDIN BINDING; LIGHT-SCATTERING; ACTIN-FILAMENTS;

D O I：

10.1016/B978-0-12-396546-2.00002-4

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：29 / 63

页数：35

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