|
||||||
|
|
|
|
|
|
|
|
Dr. Michael Bruno Post-doc michael.bruno@yale.edu 
Fluorescence Studies of NS3 helicase translocation from Hepatitis C virus The NS3 helicase, which is essential for Hepatitis C viral replication, is a member of the DExH/D proteins that constitute a subgroup of the Superfamily 2 helicases. Proteins from this superfamily are involved in all aspects of RNA metabolism, from transcription to RNA splicing as well as viral replication. The NS3 helicase is a processive molecular motor that can unwind both RNA and DNA duplexes (1). It does so by utilizing the energy of NTP hydrolysis to translocate along its nucleic acid substrate. Previous study has shown how RNA duplex separation is achieved by periodic cycles of unwinding and pausing, defining the physical and kinetic step size for NS3 as 18bp (2). However the precise mechanism by which NS3 translocates remains unclear as does the manner in which it may act differently on DNA. Several lines of evidence have also supported the hypothesis that NS3 functions as a dimer rather than monomer, however as yet there is no direct evidence for this. Thus, although previous efforts have resolved some interesting aspects of how the NS3 helicase operates, some important questions remain unanswered. By employing fluorescence techniques to study NS3 translocation, we hope to resolve those aspects of NS3 mechanism that cannot be better examined using other approaches. Fluoresence resonance energy transfer can be used to monitor duplex unwinding in solution by steady-state spectroscopy but also allows the acquisition of fast kinetic data using a stopped-flow analyzer. Additionally, specific labeling of the NS3 helicase with a fluorescent dye can be used in combination with dye labeled nucleic acid substrate to monitor translocation more directly. The FRET technique can also be applied to determine the oligomeric state of the functional helicase by using a mix of donor and acceptor dye labeled NS3. Lastly, single molecule fluorescence can be used to overcome some limitations of these approaches when faced with asynchronous or heterogeneous populations. We aim therefore, to gain a more detailed picture of how this helicase translocates which will shed greater light on its important biological role and in the process, we will develop the tools necessary to study other helicases involved in different aspects of nucleic acid metabolism. 1. Pang, P.S., Jankowsky, E., Planet, P.J. and Pyle, A.M. (2002) EMBO J. 21, 1168-76. 2. Serebrov, V. and Pyle, A.M. (2004) Nature 430, 476-80. |
|
