Interactions between RNA polymerase and the "core recognition element" are a determinant of transcription start site selection

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ~13 base pairs of promoter DNA, forming an RNAP-promoter open complex containing a single-stranded "transcription bubble," and selects a template-strand nucleotide to serve as the transcription start site (TSS). In the RNAP-promoter open complex, RNAP core enzyme makes sequence-specific protein-DNA interactions with the downstream part of the non-template strand of the transcription-bubble ("core recognition element," CRE). Here, we investigated whether sequence-specific RNAP-CRE interactions affect TSS selection. To do this, we used two next-generation-sequencing-based approaches to compare the TSS profile of wild-type RNAP to the TSS profile of an RNAP derivative defective in sequence-specific RNAP-CRE interaction. First, using massively systematic transcript end readout (MASTER), we assessed effects of RNAP-CRE interactions on TSS selection in vitro and in vivo for a library of 47 (~16,000) consensus promoters containing different TSS-region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP-CRE interaction differed from the TSS profile of wild-type RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5' merodiploid native-elongating-transcript sequencing (5' mNET-seq), we assessed effects of RNAP-CRE interactions in vivo at natural promoters in the Escherichia coli genome, and we identified 39 E. coli promoters at which RNAP-CRE interactions determine TSS selection. Our findings establish that RNAP-CRE interactions are a functional determinant of TSS selection. We propose that RNAP-CRE interactions modulate the position of the downstream end of the transcription bubble in the RNAP-promoter open complex and thereby modulate TSS selection, which involves transcription-bubble expansion or transcription bubble contraction (“scrunching” or “anti-scrunching”).