This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are studying the 3d structures of a class of RNA sequences called internal ribosomal entry sites (IRESes). These sequences are able to initiate protein synthesis in a eukaryotic cell using a mechanism that is very different from that used by the vast majority of cellular messenger RNAs. These RNAs are able to functionally substitute for a number of important proteins. We want to understand how these RNAs fold, what their 3d conformations are, and how they are able to recruit and activate the translational apparatus. A number of viruses including hepatitis A and C, poliovirus and footh & mouth disease use an IRES RNA to drive synthesis of the viral proteins. In J. Doudna's lab (Yale Univ.) we studied the IRES RNA from the hepatitis C virus (HCV). Using a variety of chemical and enzymatic probing experiments, we found strong evidence that the HCV IRES adopts a metal-ion dependent fold that can best be described as extended. We supported this hypothesis using SAXS data generated on a home setup. To see if addition of magnesium to the RNA would induce changes in the Dmax and Rg as determined by SAXS, we used an RNA known to fold into a compact shape as a control; measurements on this RNA showed a change in the Dmax 140-100 and a change in the Rg from 40-30 consistent with the known crystal structure. When we conducted the experiment on the HCV IRES, we found little or no change in the scattering, consistent with formation of a structure that remains extended in the presence of magnesium. SAXS was useful for verifying our data that the HCV IRES does to form a compact, globular fold. My lab has started work on another IRES RNA, from the plautia stale intestinal virus (PSIV). Our preliminary probing experiments show that unlike HCV, this IRES RN RNA folds into a compact, more globular structure in the presence of solvated cations. The formation of this globular structure is likely to be accompanied by a collapse of the RNA into a size and shape very different from the unfolded shape.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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Stanford University
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