RNA helicases of the DexH/D family play an essential role in viral replication and cellular RNA metabolism, including central functions in RNA splicing, translation and regulation of gene expression. Despite the importance of these proteins, their RNA helicase activity has not been subjected to enzymological study. Basic knowledge of cellular metabolism is therefore constrained by our limited understanding of reaction mechanism by motor proteins in the RNA helicase family. To address this problem, mechanistic studies have been initiated on two viral DexH/D proteins: NPH-II from Vaccinia and NS3-4A from Hepatitis C Virus (HCV). The NPH-II protein is show to be a processive, directional RNA helicase with specific roles for both the binding and hydrolysis of ATP. Having established qualitative features of NPH-II activity, this proposal aims to use direct and stopped flow kinetic measurements to determine the quantitative kinetic parameters such as translocation rates, reaction step size, processivity, helicase binding, ATP binding and hydrolytic rate constants that describe the framework for catalytic activity of this prototypical RNA helicase. In addition, the determinants for molecular recognition between RNA and helicase will be established. To determine if these findings are general and to extend the helicase studies to a viral system that poses a grave threat to public health, a complementary mechanistic framework will be developed for the HCV protein NS3-4A. This helicase will also be the subject of biophysical analyses to establish the link between cycles of ATP hydrolysis and translocative steps of the helicase protein. The mechanistic information will facilitate meaningful studies on HCV inhibitors and antiviral therapies and, given the availability of a crystal structure will set the stage for structure/function work on mutants of the NS3-4A protein. The NS3-4A protein is also useful because it is a promising candidate for novel mechanistic studies on helicase function in membrane-bound states and in the context of complex macromolecular machines.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM060620-04
Application #
6709880
Study Section
Biochemistry Study Section (BIO)
Program Officer
Ikeda, Richard A
Project Start
2000-09-01
Project End
2004-08-31
Budget Start
2003-01-01
Budget End
2003-08-31
Support Year
4
Fiscal Year
2002
Total Cost
$186,921
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Taylor, Sean David; Solem, Amanda; Kawaoka, Jane et al. (2010) The NPH-II helicase displays efficient DNA x RNA helicase activity and a pronounced purine sequence bias. J Biol Chem 285:11692-703
Serebrov, Victor; Beran, Rudolf K F; Pyle, Anna Marie (2009) Establishing a mechanistic basis for the large kinetic steps of the NS3 helicase. J Biol Chem 284:2512-21
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Beran, Rudolf K F; Pyle, Anna Marie (2008) Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase. J Biol Chem 283:29929-37
Beran, Rudolf K F; Serebrov, Victor; Pyle, Anna Marie (2007) The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate. J Biol Chem 282:34913-20
Lindenbach, Brett D; Pragai, Bela M; Montserret, Roland et al. (2007) The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication. J Virol 81:8905-18
Myong, Sua; Bruno, Michael M; Pyle, Anna M et al. (2007) Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase. Science 317:513-6
Beran, Rudolf K F; Bruno, Michael M; Bowers, Heath A et al. (2006) Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track. J Mol Biol 358:974-82
Kawaoka, Jane; Pyle, Anna Marie (2005) Choosing between DNA and RNA: the polymer specificity of RNA helicase NPH-II. Nucleic Acids Res 33:644-9
Kawaoka, Jane; Jankowsky, Eckhard; Pyle, Anna Marie (2004) Backbone tracking by the SF2 helicase NPH-II. Nat Struct Mol Biol 11:526-30

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