Poster abstracts
Poster number 43 submitted by Ila Marathe
Dissecting structural and functional cooperation in RNase P, a multi-subunit ribonucleoprotein
Ila Marathe (Department of Microbiology, Ohio State University), Yi Luo (Department of Physics, Ohio State University), Michael Poirier (Department of Physics, Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, Ohio State University)
Abstract:
RNase P is an essential ribozyme that catalyzes Mg2+-aided maturation of tRNAs in all life forms. An RNase P RNA (RPR) and five RNase P proteins (RPPs) make up archaeal RNase P. In addition to serving as a model to uncover the cooperation between a catalytic RNA and multiple protein subunits, archaeal RNase P is an excellent proxy for the intractable and homologous eukaryotic cousin whose dysregulation leads to disease. Towards our goal of understanding how proteins modulate RNA catalysis, we focus here on L7Ae, an archaeal ribosomal protein that doubles up as an RPP. L7Ae binds the kink-turn, a widespread structural motif, and causes axial bending of RNAs. To test the hypothesis that L7Ae might aid in recruiting other RPPs to the RPR at physiological [Mg2+], we seek to uncover its roles in assembly and catalysis of archaeal RNase P. Through ensemble/single-molecule fluorescence measurements and activity assays, we expect to gain insights into: (i) stoichiometry of L7Ae, (ii) influence of L7Ae on the RNase P assembly landscape, and (iii) the ability of L7Ae to modulate the Mg2+ dependence of RNase P activity. Results from our fluorescence polarization (FP) assays suggest that L7Ae indeed enhances the binding affinity of a binary RPP complex for the RPR. To parse the basis (association/dissociation) for this gain, we are exploiting single-molecule total internal reflection fluorescence spectroscopy for co-localization analyses of an immobilized Cy5-labeled RPR and soluble Cy3-labeled L7Ae. Our promising data suggest the feasibility of these methods to map the hierarchy and cooperation during assembly of archaeal RNase P. Our results are expected to shed light on how RPPs affect the structure and function of the RPR, and more broadly help elucidate the basis of human diseases where RNA-protein cooperation is defective.
Keywords: RNase P, RNA-protein cooperation, L7Ae