Transcription is a fundamental catalytic process in which RNA is produced on a linear DNA template. The process is processive which means that once the RNA polymerase initiates its journey down the DNA it does not leave the template DNA molecule until the RNA molecule being synthesized is complete. In order to satisfy the topological constraints inherent to this process, it is believed that there must be a relative rotation of the DNA template and the RNA polymerase, much as the relative rotation of a nut and bolt during tightening. This proposal is devoted to experiments which will allow for a quantitative measurement of the importance of this rotation during transcription. The long term aims of this proposal are the complete understanding of the dynamic and structurally detailed events associated with transcription.
The specific aims for the immediate new funding period include the successful completion of three independent experiments which relate to the structure of the transcription complex. In the first experiment, a circular DNA template is tethered so that it is no longer able to rotate freely about its helix axis and the effects of this tether on the kinetics of transcription are measured. In the second approach, fluorescence correlation spectroscopy will be developed as a tool for measuring DNA rotation and then will be applied to this problem. The third experiment will probe detailed nucleic acid base pairing in the ternary complex of DNA, RNA, and RNA polymerase by site specific psoralen photo-crosslinkage. These experiments relate in a fundamental way to the regulation of genes in chromosomes, for the in vivo activity of large regions of chromosomes in transcription may well be regulated by the freedom of that region of the DNA to rotate freely about its helix axis. The long term goal is to apply the fluorescence correlation spectroscopy technique to template DNA inside cells. If perfected, the dynamics of in vivo transcription will be measureable inside cells.
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