Exploring the translocation mechanism of transcription termination factor rho.

摘要

In prokaryotic organisms there are two forms of transcription termination, factor independent and factor dependent. Rho is a hexameric transcription termination factor isolated from E coli. The protein requires a bound cofactor, polynucleotide, and substrate, ATP, in order to terminate transcription. Termination activity is accompanied by observed hydrolysis of substrate, and helicase activity involving the separation of the RNA-DNA hybrid duplex.; This study determined the number of nucleotide substrate binding sites on each rho hexamer, and the correlation between available and competent nucleotide binding sites and rho activities. The determination of the parameters effecting NTP binding was carried out throughout the use of a novel photoactive ATP analog 4-thiouracil triphosphate. When radiolabeled the analog allowed determination of the exact number of NTP binding sites occupied and this is correlated with the level of NTPase activity present in that sample.; Additionally, the kinetic constants for the binding and release of both substrate and cofactor in the pre hydrolysis state were made. These measurements were carried out through the use of fluorescent labeled substrate and cofactor. The determination of these rates for binding and release allow for further support of past observations and the presentation of a tethered tracking mode of rho motion along the nascent RNA transcript. The tethered tracking model for the motion of rho is supported by previous observations, and the stoichiometry of cofactor release has determined that a single RNA binding site on each rho dimer is released. This single release supports only a tethered tracking mechanism of rho motion.; These above studies in conjunction with previous experimentation allow for the determination of the order of events in the kinetic hydrolytic cycle and support the previously proposed functional dimer model for rho. This work also helps in the understanding of the motion of rho along the nascent transcript. Transcription termination factor rho is a good model system for the study of the action of a molecular motor and will add information to the expanding field of energy transduction and the relationship between structure and function.