Post-translational modification of histones plays an integral role in regulation of genomic expression through modulation of chromatin structure and function. Chemical preparations of histones bearing these modifications allows for comprehensive in vitro mechanistic investigation into their action to deconvolute observations from genome-wide studies in vivo. Previously, we reported the semisynthesis of ubiquitylated histone H2B (uH2B) using two orthogonal expressed protein ligation reactions. Semisynthetic uH2B, when incorporated into nucleosomes, directly stimulates methylation of histone H3 lysine 79 (K79) by the methyltransferase, disruptor of telomeric silencing-like (Dot1L). Although recruitment of Dot1L to the nucleosomal surface by uH2B could be excluded, comprehensive mechanistic analysis was precluded by systematic limitations in the ability to generate uH2B in large scale. Here we report a highly optimized synthesis of ubiquitylated H2B bearing a G76A point mutation u(G76A)H2B, yielding tens of milligrams of ubiquitylated protein. u(G76A)H2B is indistinguishable from the native uH2B by Dot1L, allowing for detailed studies of the resultant trans-histone crosstalk. Kinetic and structure-activity relationship analyses using u(G76A)H2B suggest a noncanonical role for ubiquitin in the enhancement of the chemical step of H3K79 methylation. Furthermore, titration of the level of uH2B within the nucleosome revealed a 1:1 stoichiometry of Dot1L activation.