Molecular mechanism of promoter opening by RNA polymerase III

RNA polymerase III (Pol III) assembles together with transcription factor IIIB (TFIIIB) on different promoter types to initiate the transcription of small, structured RNAs. Here, we present structures of Pol III pre-initiation complexes comprising the 17-subunit Pol III and hetero-trimeric transcription factor TFIIIB with subunits TATA-binding protein (TBP), B-related factor 1 (Brf1) and B double prime 1 (Bdp1) bound to a natural promoter in different functional states. Electron cryo-microscopy (cryo-EM) reconstructions varying from 3.7 Å to 5.5 Å resolution include two early intermediates in which the DNA duplex is closed, an open DNA complex and an initially transcribing complex with RNA in the active site. Our structures reveal an extremely tight and multivalent interaction of TFIIIB with promoter DNA and explain how TFIIIB recruits Pol III. TFIIIB and Pol III subunit C37 together activate the intrinsic transcription factor-like activity of the Pol III-specific heterotrimer to initiate melting of double-stranded DNA in a mechanism similar as used in the Pol II system.