ABSTRACT
The master tumor suppressor p53 activates transcription in response to various cellular stresses in part by facilitating recruitment of the transcription machinery to DNA. Recent studies have documented a direct yet poorly characterized interaction between p53 and RNA polymerase II (Pol II). Therefore, we dissected the human p53/Pol II interaction via single-particle cryo-electron microscopy, structural docking, and biochemical analyses. This study reveals that p53 binds Pol II via the Rpb1 and Rpb2 subunits, bridging the DNA-binding cleft of Pol II proximal to the upstream DNA entry site. In addition, the key DNA-binding surface of p53, frequently disrupted in various cancers, remains exposed within the assembly. Furthermore, the p53/Pol II cocomplex displays a closed conformation as defined by the position of the Pol II clamp domain. Notably, the interaction of p53 and Pol II leads to increased Pol II elongation activity. These findings indicate that p53 may structurally regulate DNA-binding functions of Pol II via the clamp domain, thereby providing insights into p53-regulated Pol II transcription.
Subject(s)
Models, Molecular , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , Cryoelectron Microscopy , Exodeoxyribonucleases/metabolism , Humans , Protein Binding , Protein Domains , Protein Structure, Quaternary , Transcription Elongation, GeneticABSTRACT
Xenophagy is a form of selective autophagy that involves the targeting and elimination of intracellular pathogens through several recognition, recruitment, and ubiquitination events. E3 ubiquitin ligases control substrate selectivity in the ubiquitination cascade; however, systematic approaches to map the role of E3 ligases in antibacterial autophagy have been lacking. We screened more than 600 putative human E3 ligases, identifying E3 ligases that are required for adaptor protein recruitment and LC3-bacteria colocalization, critical steps in antibacterial autophagy. An unbiased informatics approach pinpointed RNF166 as a key gene that interacts with the autophagy network and controls the recruitment of ubiquitin as well as the autophagy adaptors p62 and NDP52 to bacteria. Mechanistic studies demonstrated that RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189. Thus, our study expands the catalog of E3 ligases that mediate antibacterial autophagy and identifies a critical role for RNF166 in this process.