Structure of the closed Pol II transcription initiation complex and implications for promoter opening

Hantsche, Merle A. (2016): Structure of the closed Pol II transcription initiation complex and implications for promoter opening. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Transcription is the first step of gene expression in all living cells. Regulatory mechanisms of transcription are fundamental for cell differentiation, organism development and adaption to environmental changes. One key event is transcription initiation. In eukaryotic cells RNA polymerase II (Pol II) transcribes messenger RNA and assembles with the general transcription factors (TF) -IIA, -IIB, -IID, -IIF, -IIE and -IIH on promoter DNA. DNA is opened and a 'transcription bubble' is formed that allows Pol II to synthesise a complementary copy of the genetic information. The molecular mechanisms of promoter assembly and opening remain poorly understood owing to the limited resolution of previous structural studies, the large size of the complex and the dynamics of the process. We report the cryo-electron microscopy structure of a closed transcription initiation complex containing Saccharomyces cerevisiae Pol II and the general transcription factors except TFIIH on double-stranded promoter DNA at 8.8 Angstrom resolution. Additionally, we show in a separate crystallographic study that the yeast-specific N-terminus of TFIIF subunit Tfg1 binds to the Pol II external 1 region. A high-resolution structure of the respective open complex at 3.6 Angstrom resolution containing a 15 nucleotide mismatch transcription bubble served for model building of the closed complex. The open complex structure reveals detailed information on the intricate interactions of the general transcription factors with each other, with Pol II and promoter DNA and it suggests a mechanism of DNA template strand loading into the Pol II active centre cleft. In the transition from closed to open complex formation we identify movements mainly in TFIIE. DNA opening occurs around the tip of the Pol II clamp and the TFIIE extended winged helix domain. In functional assays we show that the TFIIE extended winged helix domain and recruitment of TFIIE through its E-ribbon domain are important for transcription in vitro and in vivo. Moreover, we report that promoter opening can occur in the absence of the ATP-dependent factor TFIIH. Analysis of the closed complex data reveals that most of the particles (72 %) resemble the open complex structure and contain open promoter DNA. Based on our data we propose a general model how promoter opening can be achieved by the use of binding energy alone. Finally, our results underline the high structural conservation between the human and yeast transcription initiation systems.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Gene transcription, transcription initiation, RNA-Polymerase II, structural biology
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 14. November 2016
• 1. Bericht­erstatter:in: Cramer, Patrick