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...

The 3’end genomic region encodes a wide range of regulatory process including mRNA stability, 3’ end processing and translation. Here, we systematically investigate the sequence determinants of 3’ end mediated expression control by measuring the effect of 13,000 designed 3’ end sequence variants on constitutive expression levels in yeast. By including a high resolution scanning mutagenesis of more than 200 native 3’ end sequences in this designed set, we found that most mutations had only a mild effect on expression, and that the vast majority (~90%) of strongly effecting mutations localized to a single positive TA-rich element, similar to a previously described 3’ end processing efficiency element, and resulted in up to ten-fold decrease in expression. Measurements of 3’ UTR lengths revealed that these mutations result in mRNAs with aberrantly long 3’UTRs, confirming the role for this element in 3’ end processing. Interestingly, we found that other sequence elements that were previously described in the literature to be part of the polyadenylation signal had a minor effect on expression. We further characterize the sequence specificities of the TA-rich element using additional synthetic 3’ end sequences and show that its activity is sensitive to single base pair mutations and strongly depends on the A/T content of the surrounding sequences. Finally, using a computational model, we show that the strength of this element in native 3’ end sequences can explain some of their measured expression variability (R = 0.41). Together, our results emphasize the importance of efficient 3’ end processing for endogenous protein levels and contribute to an improved understanding of the sequence elements involved in this process.

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Posttranslational modifications (PTMs) of proteins are an integral part of major cellular regulatory mechanisms dictating protein function, localization, and stability. The capacity to screen PTMs using protein microarrays has advanced our ability to identify their targets and regulatory role. This chapter discusses a unique procedure that combines functional extract-based activity assay with large-scale screening utilities of protein microarrays. This "PTM-profiling" system offers advantages in quantitatively identifying modifications in an unbiased manner in the context of specific cellular conditions. While the possibilities of studying PTMs in different settings are enormous, the immune system presents an attractive model for studying the effects of perturbations in PTMs, and specifically the ubiquitin system, as these were already implicated in both immune function and dysfunction. This chapter discusses the significance of PTM profiling in addressing basic questions in immunology. We describe detailed protocols for the preparation of functional cell extracts from immune cell cultures, following differentiation or induced signals, and screening PTMs on protein arrays, as well as basic guidelines for data analysis and interpretation.

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