Photocleavage of DNA and photofootprinting of E. coli RNA polymerase bound to promoter DNA by azido-9-acridinylamines. |
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| Authors: | C Jeppesen O Buchardt U Henriksen and P E Nielsen |
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| Affiliation: | Department of Biochemistry B, Panum Institute, University of Copenhagen, Denmark. |
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| Abstract: | The long-wavelength ultraviolet (lambda approximately 420 nm) radiation induced reaction between 6-azido-2-methoxy-9-acridinylamines and supercoiled plasmid DNA results in single strand scissions and formation of covalent adducts (ratio approximately 1:10). By treating azidoacridine-photomodified DNA with piperidine at 90 degrees C, additional strand scissions are observed in a complex sequence dependent manner with an overall preference for T greater than or equal to G greater than C much greater than A. The resulting DNA fragments migrate as 5'-phosphates in polyacrylamide gels. Photofootprinting of the binding site of RNA-polymerase on promoter DNA is demonstrated with an azido-9-acridinylamino-octamethylene-9-aminoacridine. Similar experiments using 9-amino-6-azido-2-methoxyacridine indicate that this reagent recognizes changes in the DNA conformation induced by RNA polymerase binding, in relation to open complex formation. |
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