Multiplexed protein-DNA crosslinking: scrunching in transcription start site selection

In bacterial transcription initiation, RNA polymerase (RNAP) selects a transcription start site (TSS) at variable distances downstream of core promoter elements. Using next-generation sequencing and unnatural-amino-acid-mediated protein-DNA crosslinking, we have determined, for a library of 4 10 promoter sequences, the TSS, the RNAP leading-edge position, and the RNAP trailing-edge position. We find that a promoter element upstream of the TSS, the “discriminator,” participates in TSS selection, and that, as the TSS changes, the RNAP leading-edge position changes, but the RNAP trailing-edge position does not change. Changes in the RNAP leading-edge position but not the RNAP trailing-edge position are a defining hallmark of the “DNA scrunching” that occurs concurrent with RNA synthesis in initial transcription. We propose that TSS selection involves DNA scrunching prior to RNA synthesis.