Poster abstracts
Poster number 110 submitted by Katelyn Perroz
Limited proteolysis of a DEAD-Box protein reveals small conformational changes
Katelyn R Perroz (Allegheny College, Department of Biochemistry), Michelle Raymond (Allegheny College, Department of Biochemistry), Megan Arnold (Allegheny College, Department of Biochemistry), Dr. Ivelitza Garcia (Allegheny College, Department of Biochemistry)
Abstract:
RNA unwinding proteins play an essential role in most RNA processing pathways. One ubiquitous family of RNA unwindases are DEAD-Box proteins. DEAD-box proteins are characterized by 12 conserved motifs and two Rec-A like structural domains. These proteins utilize the conformational changes that occur during ATP hydrolysis to modulations in RNA-RNA, RNA-protein, and protein-protein interactions. For example, Rok1p is a yeast DEAD-Box protein involved in rRNA processing. Rok1p’s function is dependent on ATP hydrolysis. Interestingly, this protein has both an N-terminal domain (NTD) and a C-terminal domain (CTD). The peripheral domains are hypothesized to play a role in rRNA processing and protein recruitment. Rrp5, an rRNA processing factor, binds pre-rRNAs and guides ITS1 cleavage separating the large subunit RNA and small subunit RNA during biogenesis. The interaction and regulation between Rrp5 and Rok1p are proposed to be controlled by Rok1p’s peripheral domains. Thus, the effects of Rok1p’s peripheral domains on the Rec-A structural dynamics were explored with domain truncations in combination with limited proteolysis. Since previous work suggests that Rok1p is more dynamic at higher temperatures as well as in the presence of certain ligands, a comparative analysis was also performed in the presence and absence of RNA under various conditions. Proteolysis patterns dramatically change in the absence of both peripheral domains, suggesting that both the NTD and CTD domain affect the global dynamics of Rok1p. Similar results were obtained in the presence of RNA. The structural dynamics are noticeably affected in the presence of the CTD and RNA at lower temperatures. Whereas, the NTD domain effects are prevalent at high temperatures. This study demonstrated the variable structural consequence of peripheral domains.
Keywords: RNA , DEAD-Box, Proteins