XACT seq comprehensively defines the promoter position and promoter sequence determinants for initial transcription pausing
Description
Our goal was to develop a method that allowed single-nucleotide mapping of the RNA polymerase active center in cells and to apply this approach to define the promoter-sequence and promoter-position determinants of initial transcription pausing. We developed an approach to define initial transcription pausing over vast sequence space that involved incorporation of a photoactivatible unnatural amino acid into a core subunit of RNA polymerase, UV irradiation to generate covalent crosslinks between the unnatural amino acid and DNA nucleotides within several angstroms, denaturing purification of covalently linked RNA polymerase-DNA complexes, primer extension mapping of the site of the covalent link, and next-generation sequencing of the primer extension products to read out the site of crosslinking on a library of up to ~4 million sequences. The data included here are full gel images, and replicates, for data shown in Figures 3, 4, 5, S2, S3, S4, and S8. All experiments involved the electrophoresis of radiolabeled products. Figure S2 and S3 show radiolabeled RNA products. Figures 3, 4, 5, S4, and S8 show radiolabeled primer extension products that map the site photocrosslinking.