Structural basis of the complete RNA polymerase I initiation apparatus

• Applicant: Professor Dr. Christoph Engel
• Subject Area: Structural Biology
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 394580547

Transcription of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is essential to all eukaryotic organisms. In contrast to Pol II and Pol III, which mainly produce messenger and transfer RNA, respectively, the Pol I system remains poorly understood at a molecular level despite its importance. Only very recent studies began to unravel the structural basis underlying the mechanisms of Pol I transcription initiation. Solving the crystal structures of Pol I itself and of the conserved initiation factors Rrn3 and Core Factor (CF) laid the groundwork, and technological advances in cryo-EM enabled us to determine the architecture of an initially transcribing complex including all three factors. This work proposed that biophysical properties rather than a specific DNA sequence may define the conserved element of the rRNA promoter, conceptually advancing mechanistic investigation and overcoming a year-long stalemate created by a lack of promoter sequence conservation. However, specifically targeting Pol I to its one promoter and enabling high native initiations rates in vivo is achieved by an entirely different complex, the Upstream Activating Factor (UAF). But these are not the only functions of UAF: In yeast, the complex also prevents rRNA transcription by Pol II. Consequently, the knock-out of UAF subunits leads to Pol II transcribing rRNA, which renders the otherwise essential Pol I obsolete. How one factor can target and enhance Pol I initiation on the one hand and achieve a silencing of Pol II transcription on the other hand remains mysterious. Furthermore, none of the six UAF subunits reveals an apparent homology to initiation factors employed by related transcription systems. Investigation of the complete Pol I initiation apparatus will thus move into uncharted territory in an explorative, yet hypothesis-driven manner. To achieve this, we will aim at a structural and functional characterization of UAF to determine its interplay with CF, the TATA-binding protein (TBP), the rRNA promoter and Pol I itself will shed light on fundamental questions. Based on bioinformatic predictions and exploratory experimental domain/subunit definition we will use a structural biology hybrid approach combining X-ray crystallography with cryo-EM and functional assays. This will enable us to determine underlying molecular mechanisms and facilitate a comparison with molecular strategies employed by Pol II and III systems. Thereby, we may be able to explain why the entire Pol I transcription system devoted exclusively to the production of a single transcript has evolved in an unprecedented manner. In addition, recent clinical developments have established targeting Pol I transcription initiation as an extraordinarily effective therapeutic strategy in the treatment of various cancer types. Thus, understanding the molecular details of the complete Pol I initiation system will serve as the basis for targeted, hypothesis-driven development of future therapeutic strategies.

• DFG Programme: Independent Junior Research Groups
• Major Instrumentation: FPLC
• Instrumentation Group: 1350 Flüssigkeits-Chromatographen (außer Aminosäureanalysatoren 317), Ionenaustauscher