Transcription termination factor Rho is an ATPase that binds to the nascent RNA transcript and catalyzes its site-specific release within E.coli and coliphage transcription units. This proposal outlines a kinetic and thermodynamic approach with the goal of gaining a greater physical understanding of the molecular interactions and dynamic processes that occur during the termination reaction. A synthetic paused ternary complex will be constructed from the E. coli rho attenuator (att) region, an important Rho-dependent terminator that is located in front to the rho gene, and which autogenously regulates the level of Rho in the bacterial cell. The use of a recent technique to generate a homogeneous population of paused transcription complexes will be used. A transcription blockade is created through the binding of a cleavage-defective EcoR1 mutant endonuclease bound to an engineered EcoR1 site placed at strategic locations within the att region. The energetics of Rho assembly and the kinetics of transcript release will be measured to test key questions about the termination reaction. The RHo binding site on the attenuator transcript will be determined by RNA footprinting studies using primer extension/chemical modification techniques. The relationship between the site of the blockade and the energetics and kinetics of Rho attachment and transcript release will be evaluated.
