Structural Studies of Translocation Mechanism and Chemomechanical Coupling in Hexameric Helicases
- Author(s): Thomsen, Nathan David
- Advisor(s): Berger, James M
- et al.
Ring-shaped oligomeric ATPases are essential for a variety of cellular processes ranging from protein and nucleic acid metabolism to organelle transport. A subset of these motor proteins, the hexameric helicases, couple the binding and hydrolysis of ATP to the physical manipulation of nucleic acids in processes such as gene regulation, DNA replication, and DNA repair. Although all known hexameric helicases belong to the P-loop ATPase protein superfamily, they have diverged into two sub-families distinguished by their opposing translocation directions along single stranded nucleic acids: the 3'-5' AAA+ and the 5'-3' RecA-like enzymes. To understand the translocation mechanism of a 5'-3' RecA-like hexameric helicase, I crystallized the Rho transcription termination factor from E. coli bound to both RNA and ADP*BeF3. After overcoming a unique case of non-merohedral twinning, I solved multiple structures of an asymmetric Rho hexamer representing potential translocation intermediates. The ligand binding states observed in the structures reveal the mechanism by which nucleic acid binding stimulates ATPase activity and how this activity is linked to nucleic acid translocation in Rho. Comparisons with the 3'-5' AAA+ hexameric helicase E1, from papillomavirus, further reveal the structural basis for translocation polarity in AAA+ and RecA-like hexameric helicases. The work presented in this dissertation helps to explain years of biochemical studies, unifies many elements of RecA-like hexameric helicase mechanism, and has implications for understanding the hexameric motor protein family as a whole.