Requirements for RNA polymerase II preinitiation complex formation in vivo

Abstract

Transcription by RNA polymerase II requires assembly of a preinitiation complex (PIC) composed of general transcription factors (GTFs) bound at the promoter. In vitro, some GTFs are essential for transcription, whereas others are not required under certain conditions. PICs are stable in the absence of nucleotide triphosphates, and subsets of GTFs can form partial PICs. By depleting individual GTFs in yeast cells, we show that all GTFs are essential for TBP binding and transcription, suggesting that partial PICs do not exist at appreciable levels in vivo. Depletion of FACT, a histone chaperone that travels with elongating Pol II, strongly reduces PIC formation and transcription. In contrast, TBP-associated factors (TAFs) contribute to transcription of most genes, but TAF-independent transcription occurs at substantial levels, preferentially at promoters containing TATA elements. PICs are absent in cells deprived of uracil, and presumably UTP, suggesting that transcriptionally inactive PICs are removed from promoters in vivo.

Kin28 depletion increases association of TFIID subunits Taf1 and Taf4 with promoters in Saccharomyces cerevisiae

“…At the same time, there is considerable overlap between the TBP/Taf ratios (and of other properties) between TATA+ and TATApromoters; the most parsimonious explanation is that either of at least two distinct mechanisms or pathways can operate at both types of promoters, but their relative efficiency differs depending on the presence of a consensus TATA element. This notion is consistent with the idea that the categorization of genes as SAGA-and TFIID-dominated reflects a continuum in terms of regulation, not a rigid dichotomy [4,5,34]. We also observed a greater increase in occupancy by Taf1 and Taf4 upon Kin28 depletion at TFIID-dominated than at SAGA-dominated promoters (Fig.…”

Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA

“…These new results differ from our prior Pol II ChIP experiments that did not detect gene specific Taf requirements (Warfield et al, 2017). However, it was recently suggested that this may be due to background issues with Pol II ChIP data (e.g., stalled or paused Pols) and that Pol II ChIP is best used for quantitation of changes in highly expressed genes (Petrenko et al, 2019). This explanation is consistent with our new findings.…”

“…Yeast cellular transcription is dominated by a small number of exceptionally highly expressed 1 genes and it was proposed that highly expressed genes are biased for Taf-independent 2 expression (Petrenko et al, 2019). We do find that the coactivator-redundant genes, while 3 spread throughout the range of expressed genes, are enriched in the top 10% of transcribed 4 genes.…”

“…Here we have investigated the genome-wide and gene-specific roles for the coactivators SAGA and TFIID. Several prior studies suggested that, while TFIID and SAGA make genome-wide contributions, transcription at nearly all genes is dominated by one or the other factor and that many highly expressed genes show only modest TFIID-dependence (Huisinga and Pugh, 2004;Petrenko et al, 2019). This view seemingly conflicts with other studies where TFIID and SAGA were found to have major genome-wide roles at most genes Bonnet et al, 2014;Warfield et al, 2017).…”

“…Pol II ChIP and/or analysis of newly synthesized mRNA found that the majority of genes showed 28 decreased transcription upon deletion or depletion of SAGA or TFIID subunits (Baptista et al, 29 2017;Warfield et al, 2017). However, the proposal that TFIID is generally required for 1 transcription was recently disputed based on anchor-away depletion of the TFIID subunit Taf1 2 (Petrenko et al, 2019). Analysis of the top 10% of transcribed genes, analyzed by Pol 3 showed that many genes in the SAGA-dominated class were only modestly sensitive to Taf1 4 depletion.…”