Poster abstracts

Poster number 40 submitted by Mike Marvin

Investigating Substrate Interactions of Yeast Nuclear RNase P

Michael C. Marvin (Biological Chemistry at University of Michigan), David R. Engelke

Abstract:
Ribonuclease P (RNase P) is an essential endoribonuclease that catalyzes the cleavage of 5´ leader sequences of precursor transfer RNAs (pre-tRNAs). The enzyme is found in all phylogenetic domains; bacteria, archaea, and eucarya. The bacterial enzyme consists of a single RNA and one small protein. However, in the yeast Saccharomyces cerevisiae, nuclear RNase P is comprised of a single RNA and nine protein subunits. Each of the nine protein subunits, and the RNA subunit, are essential and it is not currently clear why the eukaryotic RNase P requires such a vast increase in its protein content. The proteins have potential functions in stabilizing RNA structure, substrate binding, substrate determination, roles in catalysis and localizing the holoenzyme.

In order to obtain information concerning the general structure of yeast RNase P we have initiated UV crosslinking studies with purified yeast nuclear RNase P holoenzyme and pre-tRNA substrate. To enable the detection of RNA-protein crosslinks and for purification purposes, nine yeast strains have been constructed where each of the individual proteins have been 6xHis tagged in parallel with the Rpr2 protein being TAP tagged. The results of this study will provide a framework for considering the overall architecture of the ribonucleoprotein and its interaction with pre-tRNA substrate.

Keywords: crosslinking, RNase P, tRNA