Molecular mechanism of Bre1-Rad6 mediated H2B ubiquitylation

Abstract

In yeast, H3K4 methylation, the hallmark of active transcription, is known to be dependent on H2B ubiquitylation. We want to understand the mechanism of H2B mono-ubiquitylation by E3 ubiquitin ligase Bre1and E2 ubiquitin conjugating enzyme Rad6. Bre1 has a coiled coil motif (CCM) at the N-terminus and a RING finger domain at the C-terminal end. Previous studies have well confirmed the Interaction between Bre1-CCM and Rad6. This interaction is non-canonical E2-E3 interaction because most of the E2 enzyme binds to the RING-finger domain of E3. By co-expressing Bre1-CCM and Rad6 in E.coli, we purified the Bre1-CCM/Rad6 complex and further identified the X-ray crystal structure. To validate the structure obtained from X-ray crystallography, we further purified various mutant proteins and observed the interaction change between two proteins and also H2B ubiquitylation activity.