To achieve faithful transcription, RNA polymerase (Pol) I, II and III rely on different sets of transcription factors, assembling into specific pre-initiation complexes (PICs) on cognate gene promoters.
However, recent studies revealed that the three eukaryotic transcription machineries share a core initiation complex, which is structurally and functionally conserved. In particular, the TATA-binding protein (TBP) is shared between RNA Pol I, Pol II and Pol III, while transcription factor IIB (TFIIB) and TFIIB-related factors Rrn7 and Brf1 (for RNA Pol I and III, respectively) display an high degree of structural and functional conservation.
RNA Pol III requires only the transcription factor TFIIIB, which is formed by Brf1, TBP and B", in order to drive specific transcription on a template containing a TATA-box upstream the transcriptional start site in vitro. This feature is unique to the RNA Pol III system and reflects the higher complexity of the enzyme's subunits composition. However, given the overall conservation of initiation core complexes when external factors are taken into account for RNA Pol I and II, the RNA Pol III PIC could represent a minimal initiation-competent core complex, whose molecular determinants are likely to be conserved also in RNA Pol I and II.
Therefore, we were able to isolate, purify and biochemically characterise a RNA Pol III PIC, composed of an intact 17-subunit endogenous yeast RNA Pol III enzyme, a recombinant TFIIIB complex and an endogenous TATA-box containing segment, based on the U6 promoter.
In order to unravel the molecular details of transcription initiation, a fundamental, highly regulated yet obscure step of the transcriptional cycle, we are now investigating the architecture of this complex by combining X-ray crystallography and cryo-EM. The combination of these two highly complementary approaches will allow us to obtain a detailed snapshot of the RNA Pol III-PIC at molecular level. This will not only contribute in the field of basic mechanisms of gene transcription and regulation but will also impact cancer biology, as RNA Pol III PIC assembly and activity is highly deregulated in many forms of cancer.