Transcription termination protein rho of E. coli acts to release newly synthesized RNA from the DNA template and from paused RNA polymerase. Rho is an ATPase which is active only when RNA is bound, and rho must hydrolyze ATP to achieve transcript release. The mechanism of RNA-dependent nucleotide hydrolysis and the role of such hydrolysis in transcript release are not known. The goal of the present research is to understand both chemical and kinetic features of ATP binding, RNA binding, and ATP hydrolysis by rho and to apply this knowledge toward an understanding of the molecular mechanism of transcript release. The proposed experiments are concerned with (1) the stoichiometry, rates, and order of events on the enzyme: how many molecules of ligand bind, how fast, and in what order; (2) the overall reaction stoichiometry: how many molecules of ATP are hydrolyzed per RNA chain released; and (3) the molecular details of interactions between the enzyme and its substrates: what are the interactions by which the substrates bind, and what amino acid residues are important in catalysis? These experiments test specific models of rho action, which will either be supported or discarded based on the results. Aspects of this work will contribute to the understanding of other proteins that bind ATP or RNA and to the more general problem of biological energy transduction. This investigator is studying the process by which proteins are synthesized by the process of translation from messenger RNA. The last step in this process is the so-called termination step in which the completed protein chain is released from the RNA. This is a step which requires energy in the form of ATP. Dr. Stitt wishes to elucidate the mechanism by which this complex reaction takes place.
