Poster abstracts

Poster number 56 submitted by Ross Wilson

Structural Studies of Archaeal RNase P

Ross Wilson (Department of Biochemistry, Ohio State University), Dileep Pulukkunat (Department of Biochemistry, Ohio State University), Venkat Gopalan (Department of Biochemistry, Ohio State University), Mark P. Foster (Department of Biochemistry, Ohio State University)

Abstract:
Ribonuclease P (RNase P) is a ubiquitous and essential ribozyme that cleaves the 5' leader sequence of precursor-tRNA to yield mature tRNA. RNase P is also a ribonucleoprotein complex, consisting of a large RNA subunit and a number of associated proteins that are necessary for activity in vivo. Although the RNA portion of the enzyme is conserved between the domains, the protein content varies greatly: one protein in bacteria; 4 or 5 proteins in archaea; and 10 proteins in eukarya. Interestingly, the archaeal proteins are homologous to a subset of the eukaryal proteins, yet neither domain contains a protein with sequence similarity to the single bacterial RNase P protein. An inverse correlation between RNA proficiency and associated protein content can be observed when comparing the domains of life: the RNA is active alone in vitro in the protein-sparse bacteria; active alone in only some archaea; and has not been shown to be active without the proteins in the protein-heavy eukaryal RNase P.

Recent efforts in our lab and others' have elucidated the three-dimensional structures of the individual protein subunits associated with archaeal RNase P, but the enticing question remains: how do these proteins confer an activity boost to the RNA? In bacteria, the single protein's role is relatively well-understood, but in archaea and eukarya the function of the multiple proteins is still unknown. It has been hypothesized that the proteins may aid substrate binding and specificity, or alter the structure of the RNA to stabilize an active conformation.

To address this question, we will investigate the structure of RNase P from Pyrococcus furiosus (Pfu). Instead of tackling the entire holoenzyme, we have begun working with a ribonucleoprotein complex comprising a truncated mutant of the RNA subunit (containing the catalytic portion of the molecule) and the proteins Pop5 and Rpp30. A new protocol has been developed to prepare large-scale amounts of this complex, and it has been confirmed to be catalytically active. Mass spectrometry will be employed to confirm the stoichiometry of the subunits within the complex, and crystallographic screens are underway.

Keywords: RNase P, ribonucleoprotein, structural biology