MCDB seminar will be from Pratik Basnet from the Kaplan lab will be presenting their research titled:
Title: Quantitative analysis of Transcription Start Site (TSS) selection in saccharomyces cerevisiae using Pol II MASTER
Friday, January 24, 2025
A219B Langley Hall
12:00 PM
During transcription, Pol II and general transcription factors (GTFs) assemble upstream of TSSs to form the pre-initiation complex (PIC). In yeast, the PIC scans directionally to select a usable TSS for initiation. While Pol II and the factors in the PIC are highly conserved, yeast's scanning mechanism for TSS selection differs from that of humans. To understand the mechanism of TSS selection in yeast and identify factors responsible for initiation, our lab has developed a massively parallel reporter assay (MASTER) to analyze 1000s of promoter variants in a single experiment. MASTER quantitatively dissects the contributions of TSS-adjacent sequences to TSS selection by examining all possible sequence changes around TSS. I am adapting MASTER to study Pol II scanning in vitro. In vivo systems can produce indirect effects when cellular conditions are altered, making attribution of consequences difficult. However, in vitro systems allow precise control over reaction conditions, enabling direct demonstration of how factors or perturbations drive specific effects. We have successfully adapted MASTER to an in vitro initiation system. Our results show that in vitro initiation reflects scanning and exhibits sequence-dependent effects on TSS selection. Furthermore, we validated that transcription initiation is sensitive to NTPs (the substrates of RNA synthesis) in vitro, corroborating published in vivo data. Additionally, using MASTER in vivo, I have evidence that environmental variables like Mn²⁺ globally alter initiation and likely do so through changes to Pol II activity, which can be tested in vitro. Finally, I investigated the role of the GTF TFIIB (encoded by SUA7 in yeast) in sequence-selective TSS selection. Our data suggest that TFIIB residues implicated in the recognition of a specific TSS position likely do not, and we will identify the important residues in future experiments. I aim to model transcription initiation in yeast and elucidate the roles of transcription factors and environmental variables in this process.