Characterization of DNA bending during transcription initiation in thelac operon by gel electrophoresis.

摘要

This thesis develops a gel electrophoresis method which allows the determination of direction, extent, and change of DNA bending in a region of DNA of interest.;The bend angle induced by CAP protein was measured by calibrating the gel mobility of DNA molecules with respect to bend angle using fragments of DNA containing sequence-induced bends from three to nine A tracts in length in the middle of the molecule with CAP bound at the end (to correct for gel retardation due to CAP protein). The bend angle of CAP protein was measured using molecules of a similar length containing CAP bound in the middle. My results indicate that the CAP bend is equivalent to 5.6 ;Finally, I used these molecules to follow changes in the CAP bend through the initiation of transcription using a gel electrophoresis assay (Straney, D. C. & Crothers, D. M. (1985). Cell 43, 449-459) to separate the intermediates in transcription initiation. My results indicate that the bend induced by CAP is maintained and increased through the formation of an open complex, and released upon progression into an initiated complex. I propose a model in which the bend induced by CAP may function in this situation to prevent the reaction from being rate-limited at the point of moving from open to initiated complexes. The bend achieves this effect by destabilizing possible CAP-polymerase contacts, or strong protein-DNA contacts, thus facilitating RNA polymerase movement into an initiating complex.;An isomeric set of DNA molecules containing a protein-induced bend (catabolite-activator protein or CAP) next to a piece of DNA that harbors a sequence-induced bend from the kinetoplast DNA (kDNA) of the parasite Leishmania tarentolae has been constructed. The phasing between the bends is shifted over one helical turn by the addition of linkers between them, thus altering the end-to-end distance on which gel mobility depends. Taking DNA bent around CAP as a standard, I conclude that overall direction of bending is towards the minor groove at the center of the A