The Mechanochemistry of Rho Transcription Termination Factor
Joshua Adelman, University of California, Berkeley
Rho is a ring-shaped hexameric motor protein that translocates along single stranded RNA and terminates transcription of certain genes in bacteria. Using six canonical ATP binding sites, it couples chemical energy from the nucleotide hydrolysis cycle to move along mRNA protruding from RNA polymerase’s active site. We combine computational modeling with data from new biochemical assays to infer the following properties of Rho: (1) all six subunits are catalytically competent and participate in the sequential hydrolysis of ATP; (2) translocation of the RNA strand through the ring is driven by the binding transition of nucleotide in the catalytic site; (3) hydrolysis is coordinated between adjacent subunits that bind RNA; (4) hydrolysis changes the affinity of a subunit for RNA; (5) release of inorganic phosphate is slow and is controlled by modulating circumferential stress in the ring. Using a numerical optimization scheme to simultaneously analyze several transient and steady state biochemical experiments, we determine a consistent set of rate constants for each step in the mechanochemical pathway. Using this model, we determine the flux distribution through the kinetic network and demonstrate that there is no single dominant pathway. This technique provides a systematic procedure with which to study the kinetic mechanisms of multimeric, multi-state allosteric enzymes.
Abstract Author(s): Joshua L. Adelman<sup>1</sup>, Jung-Chi Liao<sup>2</sup>, George Oster<sup>2</sup><br /> <sup>