Making and Breaking Ubiquitin

Abstract

A complex interplay between DNA, histones, and chromatin associated proteins coordinates the structure and function of the eukaryotic genome. To help accomplish this task, extensive posttranslational modifications of the core histone proteins have evolved. One of the less elucidated modifications, monoubiquitylation of H2B (uH2B), has been implicated in transcription elongation and histone methylation of H3 K4 and K79. Characterization of the role of uH2B in these processes requires the isolation or generation of homogenously ubiquitylated H2B. To this end, we have developed a traceless semisynthetic strategy to generate uH2B. We employ two expressed protein ligation (EPL) reactions and two methods for ligation site removal to synthesize native uH2B. The resulting uH2B was reconstituted into core histone octamers and nucleosome arrays were then generated for biochemical characterization.Controlling protein function through posttranslational manipulations has emerged as an attractive complimentary technology to existing genetic systems. We have developed a new posttranslational, small molecule‐mediated, technology for the manipulation of protein function. This system, termed SURF (Split‐Ubiquitin for the Rescue of Function), places the complementation of ubiquitin under the control of a small molecule. Before complementation a protein of interest is targeted for destruction by the proteasome through the introduction of an N‐terminal degron. Small molecule induced dimerization results in ubiquitin complementation and folding followed by cleavage of the protein of interest, thereby releasing it from the degron and rescuing its function. This system has been successfully applied to activate several classes of proteins in living cells. This general strategy should allow for inducible rescue of a variety of proteins in such a way that their native structure and function are maintained.