Structure and Mechanism of Eukaryotic Transcription Regulation

Transcription is the first step to read out the genetic information stored in the chromosome. In eukaryotes, three multi-subunit RNA polymerases (Pols), Pol I, II, and III transcribe the 25S ribosomal RNA (rRNA), protein-coding, and short non-coding RNAs such as transfer RNA (tRNA) and 25S rRNA genes, respectively. A wealth of genetic and biochemical data accumulated over 50 years have uncovered most of the molecular players involved and details of the intricate regulatory circuits. The recruitment and assembly of the initiation complex at the promoter represent one of the key regulatory steps during gene expression. The Pol II initiation machinery includes the activator-bound Mediator along with a series of general transcription factors (GTFs) (TFIID, TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH) that assemble into a ~4-megadalton (MDa) pre-initiation complex (PIC) on core promoter DNA. On the other hand, the Pol III enzyme is recruited onto its promoter with the help of corresponding GTFs (TFIIIC, TBP, BRF1, and B’’). This proposal aims to investigate the mechanism of transcription regulation by directly visualizing these transcriptional competent initiation complexes using single-particle cryo-EM. Comparable pictures of these different but evolutionarily related systems at this critical step of gene regulation will provide an unprecedented, comprehensive view of this key process in the central dogma of molecular biology.