Inherent asymmetry of Rpd3S coordinates its nucleosome engagement and association with elongating RNA polymerase II.

The Rpd3S histone deacetylase complex has a crucial role in genomic integrity by deacetylating transcribed nucleosomes following RNA polymerase (Pol) II passage. Cryo-EM studies highlight the importance of asymmetrical Rco1-Eaf3 dimers in nucleosome binding, yet the interaction dynamics with nucleosomal substrates alongside elongating Pol II are poorly understood. Here we demonstrate the essential function of the Rco1 N-terminal intrinsically disordered region (IDR) in modulating Pol II association, in which K/R mutations within the Rco1 IDR impair interaction of Rpd3S with the C-terminal domain (CTD) of Rpb1, without affecting nucleosome recognition or complex integrity. We also identify the Rco1-PHD1 and Eaf3-CHD domains as crucial for specific binding to Ser5-phosphorylated CTD. The Rco1 IDR alleviates autoinhibition from its C terminus, facilitating PHD1-CHD engagement with phosphorylated CTD. Furthermore, we reveal a conserved mechanism by which asymmetrical Rco1-Eaf3 dimers coordinate nucleosome engagement and Pol II interaction, enhancing understanding of epigenetic complexes associated with transcriptional machinery.