Project Summary Current organization of the “Tree of Life” arranges organisms in to three domains: Bacteria, Archaea, and Eukaryota. Eukaryotes package genomes that are typically orders of magnitude larger than both prokaryotic domains by utilizing histone proteins to form nucleosomes. Originally, histones were only observed in eukaryotes, but studies over the past two decades have identified a plethora of histone sequences in many archaeal species and that these histones can package DNA with “nucleosome-like” constructs, which we call “archaeasomes”. Futhermore, some histone sequences encoded in the Asgard superphylum of Archaea have been identified to contain disordered and positively charged histone tails, a trait rarely observed in archaea but predominant in eukaryotes. These observations provided evidence for the hypothesis that Archaea, and specifically Asgards, bridge the evolutionary gap between prokaryotic and eukaryotic life. However, little is known about the solution structure of archaeasome constructs and how they regulate gene expression in the cell, and even less is understood about how different histone features, such as highly charged tails, contribute to archaeasome stability and influenced archaeasome-to-nucleosome evolution. In this proposal, we aim to characterize the inherent structure-dynamics-function relationship between archaeasome stability and cellular behaviors, such as growth rates and transcription regulation. Furthermore, we will characterize the chromatin-forming ability of putative Asgard histones and shed light on how disordered and positively charged tails aide in regulating the formation of elongated archaeasome complexes. In the first aim, we will combine computational models with in vitro biophysical characterization techniques, namely cryoEM and analytical ultracentrifuation (AUC), to determine archaeasome structures of varying sizes. We also identify key sites within archaeal histone proteins that may be mutated to allow for the formation of larger archaeasome constructs, and we will collaborate with cell biologists to correlate in vitro stabilization with in vivo transcription regulation and cell growth. For our second aim, Asgard species cannot currently be cultured, so we will utilize the in vitro methods of cryoEM, MNase digestion, and AUC to characterize the solution structure and accessibility dynamics of chromatin formed by Asgard histones, which are hypothesized to be the closest known ancestor to eukaryotic histones. We will express Asgard histones with and without the highly charged tails to determine the effect of tail sequence on chromatin structure and dynamics and infer their role in archaeasome-to-nucleosome evolution.