Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes

Retrotransposons are endogenous elements that have the ability to mobilise their DNA and integrate at different locations in the host genome. In budding yeast, the Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase III-transcribed genes, such as the genes of transfer RNAs, representing a paradigm for specific targeted integration. Here we present the cryo-EM reconstruction at 4.0 [A]-resolution of an active Ty3 strand-transfer complex (Ty3 intasome) caught in the act of integrating onto a specific tRNA gene bound to the RNA Polymerase III general transcription factor TFIIIB, which is required for Ty3 specific targeting. The structure unravels the molecular mechanisms underlying Ty3 integration specificity at RNA Polymerase III-transcribed genes and sheds light into the architecture of a retrotransposon integration machinery during the process of strand transfer at a genomic locus. The Ty3 intasome establishes contacts with a region of the TATA-binding protein (TBP), a subunit of TFIIIB, which is blocked by the ubiquitous transcription regulator negative cofactor 2 (NC2) in RNA Pol II-transcribed genes. A previously unrecognised chromodomain of the Ty3 integrase mediates non- canonical interactions with TFIIIB and the tRNA gene itself, defining with extreme precision the position of the integration site. Surprisingly, Ty3 retrotransposon tethering to TFIIIB topologically resembles LEDGF/p75 transcription factor targeting by HIV retrovirus, highlighting mechanisms of convergent evolution by unrelated mobile elements and host organisms. The Ty3 intasome-TFIIIB-tRNA promoter complex presented here represents a detailed molecular snapshot of a general transcription factors co-option by a mobile element, resulting in harmless integration into the host genome.