Requirements for RNA polymerase II preinitiation complex formation in vivo.

Natalia Petrenko,Koon Ho Wong,Liguo Dong,Kevin Struhl,Yi Jin

doi:10.7554/elife.43654

Natalia Petrenko, Koon Ho Wong + Show 3 more

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https://doi.org/10.7554/elife.43654

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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.

Highlights

Transcription by RNA polymerase (Pol) II requires assembly of a preinitiation complex (PIC) composed of general transcription factors (GTFs) bound at the core promoter (Conaway and Conaway, 1993; Buratowski, 1994; Orphanides et al, 1996; Roeder, 1996)
To systematically study the role of GTFs and Pol II on transcription, we constructed anchor-away strains for subunits of TATA-binding protein (TBP) (Spt15), TFIIA (Toa1 and Toa2), TFIIB (Sua7), TFIIE (Tfa1 and Tfa2), TFIIF (Tfg1), TFIIH (Ssl1 and Ssl2), and Pol II (Rpb1)
All GTFs including TFIIA are generally required for pol II transcription in vivo GTFs were defined originally as factors required for ‘basal’ transcription in vitro, some GTFs are dispensable or only stimulatory under certain reaction conditions

Summary

Introduction

Transcription by RNA polymerase (Pol) II requires assembly of a preinitiation complex (PIC) composed of general transcription factors (GTFs) bound at the core promoter (Conaway and Conaway, 1993; Buratowski, 1994; Orphanides et al, 1996; Roeder, 1996). GTFs were defined originally as factors necessary and sufficient for ‘basal’ transcription from core promoters in vitro. Such in vitro reactions varied with respect to the promoter used, the concentration and purity of GTFs, and the concentration and nature (e.g. supercoiled, circular, or linear) of the DNA template. TFIIE, TFIIF, and TFIIH can be dispensable or only stimulatory for transcription from negatively-supercoiled templates, depending on the promoter (Parvin and Sharp, 1993; Goodrich and Tjian, 1994; Parvin et al, 1994; Timmers, 1994). In addition to the classically defined GTFs, TBPassociated factors (TAFs) in the TFIID complex and the Mediator complex are important or required for Pol II transcription in vitro, depending on the promoter and conditions (Tjian and Maniatis, 1994; Takagi and Kornberg, 2006)

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