DNA damage triggers polyubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a “mechanism of last resort” employed during transcription stress. In yeast, this process is dependent on Def1 through a previously unresolved mechanism. Here, we report that Def1 becomes activated through ubiquitylation- and proteasome-dependent processing. Def1 processing results in the removal of a domain promoting cytoplasmic localization, resulting in nuclear accumulation of the clipped protein. Nuclear Def1 then binds RNAPII, utilizing a ubiquitin-binding domain to recruit the Elongin-Cullin E3 ligase complex via a ubiquitin-homology domain in the Ela1 protein. This facilitates polyubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Together, these results outline the multistep mechanism of Rpb1 polyubiquitylation triggered by transcription stress and uncover the key role played by Def1 as a facilitator of Elongin-Cullin ubiquitin ligase function. Display omitted •Def1 protein is proteolytically processed in response to transcription stress•Processing is ubiquitin and proteasome dependent•Processed Def1 (pr-Def1) accumulates in the nucleus, where it binds RNAPII•Using its CUE domain, pr-Def1 then recruits Elongin-Cullin via Ela1’s UbH domain DNA damage induces proteasome-dependent proteolytic processing of Def1, releasing an activated protein fragment, which highlights how irreversible proteasome-mediated cleavage of a protein can act as a posttranslational modification in this signaling pathway.