RNA - protein interactions constitute a vital part of numerous biochemical processes. In the HIV life cycle, the interaction of the viral protein Rev and the Rev Responsive Element (RRE), a part of unspliced HIV RNA, is crucial for the propagation of infectious virions. Intervention of this interaction disrupts the viral life cycle. Rev - RRE interaction initially occurs at a high affinity binding site localized to a relatively small stem loop structure called RREIIB. This binding event has been well characterized by a variety of biochemical, enzymatic and structural studies. Our collaborators have previously demonstrated the efficacy of zinc finger proteins, generated by phage display, in the specific targeting of RREIIB. We have shown that the binding of these zinc finger proteins is restricted to the bulge in stem loop IIB that Rev also targets. Currently these proteins bind RREIIB with dissociation constants in the nanomolar range. We have employed a wide assortment of biophysical techniques such as gel shift assays, circular dichroism, isothermal titration calorimetry and NMR structural studies to further investigate this interaction. Several mutants of the zinc finger protein and the RNA were also studied to delineate the parts of the protein secondary structure as well as the role of specific side chains in this interaction. We have generated a solution structure of the RREIIBTR RNA bound zinc finger protein, ZNF29G29R, which displayed the highest affinity to this RNA. This has allowed us to shed further light on the molecular basis of this RNA - protein interaction and provides input for further refinement in our structure guided phage display.
| Identifer | oai:union.ndltd.org:GEORGIA/oai:digitalarchive.gsu.edu:chemistry_diss-1021 |
| Date | 01 July 2008 |
| Creators | Mishra, Subrata H |
| Publisher | Digital Archive @ GSU |
| Source Sets | Georgia State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Chemistry Dissertations |
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