Core promoter sequence in yeast is a major determinant of expression level

Abstract

The core promoter is the regulatory sequence to which RNA polymerase is recruited and where it acts to initiate transcription. Here, we present the first comprehensive study of yeast core promoters, providing massively parallel measurements of core promoter activity and of TSS locations and relative usage for thousands of native and designed sequences. We found core promoter activity to be highly correlated to the activity of the entire promoter and that sequence variation in different core promoter regions substantially tunes its activity in a predictable way. We also show that location, orientation, and flanking bases critically affect TATA element function, that transcription initiation in highly active core promoters is focused within a narrow region, that poly(dA:dT) orientation has a functional consequence at the 3 ′ end of promoters, and that orthologous core promoters across yeast species have conserved activities. Our results demonstrate the importance of core promoters in the quantitative study of gene regulation.[Supplemental material is available for this article.]The RNA polymerase II (Pol II) core promoter is the region to which Pol II and its accompanying general transcription factors are recruited to the DNA, form the pre-initiation complex (PIC), and act to initiate transcription (Smale and Kadonaga 2003). In yeast, the PIC is recruited to a TATA element, either a consensus TATA box or a weaker one with 1-2 mismatches to the consensus (Singer et al. 1990;Basehoar et al. 2004;Sugihara et al. 2011;Rhee and Pugh 2012). In both yeast and metazoans, PIC recruitment to a TATA element leads to promoter DNA melting ∼20 base pairs (bp) downstream from it (Giardina and Lis 1993), with the promoter sequence ∼30 bp downstream from the TATA element located at the Pol II active center (Bushnell et al. 2004;Miller and Hahn 2006). While in metazoans this leads to transcription initiation ∼30 bp downstream from the TATA element, in S. cerevisiae, Pol II performs a downstream scan of the template strand in search of transcription start sites (TSSs) (Giardina and Lis 1993;Kuehner and Brow 2006;Sugihara et al. 2011;Fishburn and Hahn 2012) in a manner that depends on the sequence around and upstream of the TSS (Hahn et al. 1985;Furter-Graves and Hall 1990;Faitar et al. 2001;Zhang and Dietrich 2005;Fishburn and Hahn 2012;Goel et al. 2012). This results in transcription initiation between 40-120 bp downstream from the TATA element and typical core promoter lengths of 100-200 bp (Smale and Kadonaga 2003;Lubliner et al. 2013). To allow access of the PIC to the DNA, core promoters typically contain a nucleosome-free region (NFR) (Field et al. 2008;Kaplan et al. 2009).In the study of regulatory sequences and their effects on expression, core promoter sequences remain relatively understudied, as most efforts are directed at transcription factor (TF) binding sites and their role in determining regulatory logic and expression levels (Levo and Segal 2014). In yeast, many core promoter-related studies revolved around the effec...

High-throughput functional comparison of promoter and enhancer activities

“…This flexibility contrasts with the demonstrated requirement for core promoter elements at some promoters (Smale and Kadonaga 2003;Mogno et al 2010;Kadonaga 2012;Lubliner et al 2015). Our results suggest instead that promoter activity at CREBBP-bound loci is best understood as a function of the specific complement of transcriptional activators that are bound (Fig.…”

Combinatorial Gene Regulation through Kinetic Control of the Transcription Cycle

“…Core promoter sequence affects the ability of activators or enhancers to activate transcription (Ohler & Wassarman 2010; Lubliner et al 2015; Butler & Kadonaga 2001), but this is not incorporated into quantitative models of transcriptional control. In a kinetic framework, the core promoter plays a key role, since TF function is necessarily interpreted through the rate-limiting steps of the promoter.…”

The Determinants of Directionality in Transcriptional Initiation

“…In vitro, isolated TATA boxes support bidirectional transcription [28] (Figure 1B, Key Figure). In yeast, the TATA box is able to promote transcription in both orientations but the orientation does affect the level of transcriptional output [29]. Inversion of asymmetric TATA boxes in reporter plasmids transfected into human cells still produced transcriptional activation of the downstream genes they regulated [30].…”