Structural basis of Ty1 integrase tethering on RNA polymerase III for targeted DNA integration

The yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. Here, we report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Unexpectedly, IN1 binding associates with insertion of subunit C11 C-terminal Zn-ribbon into the Pol III funnel, which provides atomic evidence for a two-metal mechanism during RNA cleavage. Moreover, unstructured regions of subunits C53 and C37 reorganize close to C11, likely explaining the connection between the C37/C53 heterodimer and C11 during transcription reinitiation. Our results suggest that IN1 binding induces a Pol III configuration that favors chromatin residence, thus improving the likelihood of Ty1 integration.