biostudies-literatureChen FNatural Science Foundation of Tianjin CityFundamental Research Funds for the Central Universities, Nankai UniversityNational Natural Science Foundation of Chinae2025291118https://www.ebi.ac.uk/biostudies/studies/S-EPMC8379938biostudies-literatureUnknown118(33)The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), composed of five core subunits Paf1, Leo1, Ctr9, Cdc73, and Rtf1, participates in all stages of transcription and is required for the Rad6/Bre1-mediated monoubiquitination of histone H2B (H2Bub). However, the molecular mechanisms underlying the contributions of the PAF1C subunits to H2Bub are not fully understood. Here, we report that Ctr9, acting as a hub, interacts with the carboxyl-terminal acidic tail of Rad6, which is required for PAF1C-induced stimulation of H2Bub. Importantly, we found that the Ras-like domain of Cdc73 has the potential to accelerate ubiquitin discharge from Rad6 and thus facilitates H2Bub, a process that might be conserved from yeast to humans. Moreover, we found that Rtf1 HProceedings of the National Academy of Sciences of the United States of AmericaBiochemical insights into Paf1 complex-induced stimulation of Rad6/Bre1-mediated H2B monoubiquitination.PMC837993831870750030/6321105220JCYBJC0132031670758Guo LLong JGe XChen FFeng WLiu BLi DFZhou HfalseBiochemical insights into Paf1 complex-induced stimulation of Rad6/Bre1-mediated H2B monoubiquitination.The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), composed of five core subunits Paf1, Leo1, Ctr9, Cdc73, and Rtf1, participates in all stages of transcription and is required for the Rad6/Bre1-mediated monoubiquitination of histone H2B (H2Bub). However, the molecular mechanisms underlying the contributions of the PAF1C subunits to H2Bub are not fully understood. Here, we report that Ctr9, acting as a hub, interacts with the carboxyl-terminal acidic tail of Rad6, which is required for PAF1C-induced stimulation of H2Bub. Importantly, we found that the Ras-like domain of Cdc73 has the potential to accelerate ubiquitin discharge from Rad6 and thus facilitates H2Bub, a process that might be conserved from yeast to humans. Moreover, we found that Rtf1 H2021-01-01T00:00:00Z2021 Aug2025-04-05T14:05:41.5Z2025-04-05T14:05:41.5ZS-EPMC83799383438531610.1073/pnas.2025291118