During transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active to an arrested state that allows for removal of DNA lesions. This transition requires site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase, a process that is facilitated by ELOF1 in an unknown way. Using cryo-EM, biochemical assays, and cell biology approaches, we show that ELOF1 serves as an adaptor to stably position UVSSA and the CSA ubiquitin ligase on arrested RNAPII, leading to ligase neddylation and activation of RNAPII ubiquitylation. In the presence of ELOF1, a TFIIS-like element in UVSSA gets ordered and extends through the RNAPII pore, thus preventing reactivation of Pol II by TFIIS. Our results provide the structural basis for RNAPII ubiquitylation and inactivation in transcription-coupled repair. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.
