RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation.
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