Photofootprinting in vivo detects transcription-dependent changes in yeast TATA boxes

Abstract

To elucidate critical steps in the transcription initiation process, we have devised a protocol for obtaining information about DNA structure and DNA-protein interactions at nucleotide level resolution from intact yeast cells. Our procedure combines the ultraviolet light 'footprinting' method developed by Becker and Wang with the 'genomic sequencing' technique described by Church and Gilbert. Using this approach we were able to detect the binding of GAL 4 protein at sites within the upstream activating sequence (UASG) previously mapped using other in vivo and in vitro foot-printing procedures. We also observed transcription-dependent changes in sensitivity of DNA to ultraviolet-induced covalent modification at several positions between the upstream activating sequence and the transcription initiation sites of the GAL 1 and GAL 10 genes. The most prominent of these changes occurs at a common site within the putative 'TATA' boxes of the two genes. Ultraviolet modification at this site is enhanced only in transcriptionally active promoters.