Mechanism of E. coli Transcription Termination Factor Rho

Temple University, Philadelphia PA

Investigators

Abstract

Transcription termination factor Rho of E. coli releases RNA from transcription complexes. Rho, an RNA-dependent ATPase, uses the energy from ATP hydrolysis to travel 5' -> 3' along RNA. This travel may constitute the basis for the RNA-DNA helicase function of Rho, which is believed to be involved in disruption of transcription complexes and release of RNA. The long-term goal is to understand, at the molecular level, how Rho works. Amino acids of Rho that have critical active site functions in RNA-dependent ATP hydrolysis have been proposed based on crystal structures and comparisons with other proteins. There are, however, multiple candidates for many of the catalytic site roles. Site-directed mutagenesis and testing of the resultant proteins will be carried out both to identify the relevant amino acids and to pinpoint their functions. The identification of conformation changes in Rho will also be analyzed by peptide-based hydrogen/deuterium amide proton exchange. These studies will reveal the locations within Rho where amide hydrogen exchange changes when substrates, products, or transition state analogs bind. The results should reveal conformation changes that are involved in ATP hydrolysis and directional travel by Rho, and will enhance the present incomplete picture of Rho-RNA interactions. Combining the results of these two experimental approaches will allow better understanding of the coordination of ATP hydrolysis with protein conformation change. Directional protein travel along nucleic acids is of fundamental importance and wide occurrence; a more complete understanding will result from this work. Training will be provided to graduate and undergraduate students during the research, and results will be presented at scientific meetings and published in scientific journals.