Contribution of promoter architecture to Pol II initiation by scanning in Saccharomyces cerevisiae

Eukaryotic protein-coding genes are transcribed by RNA polymerase II (Pol II), which is highly conserved in structure. As the first step of transcription, initiation determines where and how efficiently transcription initiates and therefore is a key component of gene expression. Pol II initiation in yeast proceeds by a proposed promoter scanning mechanism, where the Pol II pre-initiation complex (PIC), comprising Pol II and initiation factors, assembles upstream of the initiation region and then scans downstream to select appropriate transcription start sites (TSSs) to initiate transcription. To understand why any individual TSS is used, we are determining how scanning is affected by or interacts with different promoter “architectures”, with architecture defined as the constellation of promoter attributes, including TSS sequence, core promoter-TSS distance, and PIC/TSS/adjacent nucleosome positioning or composition, etc. We have systematically designed and constructed controlled promoter variant libraries where we vary promoter attributes in a controlled fashion. Promoter libraries with all possible DNA sequence variants within a specified TSS region allow us to determine the spectrum of initiation site efficiencies. Libraries with altered core promoter-TSS distances, UAS identity, scanning region sequence composition will allow us to determine how these features contribute to initiation efficiency. By combining data from these libraries, our goal is to quantitatively model and predict Pol II initiation distributions for any particular promoter.