Poster abstracts
Poster number 117 submitted by Michelle Raymond
Structural characterization of DEAD-Box protein peripheral domains
Michelle Raymond (Biochemistry Department Allegheny College), Katie Perroz (Biochemistry Department Allegheny College), Megan Arnold (Biochemistry Department Allegheny College), Dr. Ivelitza Garcia (Biochemistry Department Allegheny College)
Abstract:
Ribosomal RNA processing and folding require many trans-acting factors to achieve mature ribosomes. DEAD-box proteins are essential for both small and large subunit formation. These proteins couple ATP hydrolysis and protein conformational changes to RNA folding and/or RNP complex modulation. For example, Rok1p, a yeast DEAD-Box protein, guides the cleavage of ITS1 in pre-rRNA to separate the large and small subunit RNAs. Structurally, Rok1p contains two Rec-A like domains the form an ATP active site and an RNA binding surface. Two peripheral domains [the N-terminal domain (NTD) and C-terminal domain (CTD)] flank the Rec-A domains and are proposed to aid in RNA and protein recruitment. However, recent studies have suggested a role in ATP binding. Thus, limited proteolysis was utilized to examination global structural changes in Rok1p and Rok1p domain truncation in the presence of ADP or AMPPNP as well as under varying temperatures. ADP binding to Rok1p and Rok1p variants yielded two predominate proteolytic fragments. The rate of cleavage was greatly affected by the binding of ADP. However, AMPPNP (nonhydrolyzable analog) binding limited proteolysis under various temperatures and peptidase concentrations. The presence of CTD affects the extent and rate of cleavage at all temperatures. In the presence of both peripheral domains, the structural stability caused by the NTD lessens the CTD median effects. This dampening of proteolysis affects ADP bound protein more than the AMPPNP bound complex. Although minor structural changes are suggested, this study shows that peripheral domains have variable structural consequences in the presence of nucleotide-binding.
Keywords: RNA, DEAD-Box, Limited Proteolysis