Elucidating the Roles of Intrinsically Disordered Proteins in Eukaryotic Transcriptional Regulation with Single-Molecule Imaging.

Over 30% of the eukaryotic proteome is comprised of intrinsically disordered protein regions (IDRs) that cannot fold into stable three-dimensional structures. Despite lacking well-defined structures, many IDRs play functional roles in critical cellular processes. One such process is transcription, where many regulatory proteins are intrinsically disordered proteins (IDPs) containing substantial IDRs. Although numerous structured proteins have functions characterized by structural biology approaches, IDRs are less amenable to structure-function relationship characterization using the same methods. Fluorescence microscopy is useful in bridging this gap as it can measure the physical behaviors of IDPs that are important for their functions, e.g., their spatial distribution and diffusion/binding dynamics in the cell. While many fluorescence microscopy methods can serve this purpose, single-molecule imaging methods that enable high spatial and molecular resolution are uniquely capable of accurately characterizing dynamic IDP interactions in transcriptional regulation. In this review, we introduce widely applied single-molecule imaging methods, summarize insights into IDP biology and transcriptional regulation that they have enabled, and discuss their caveats, limitations, and potential directions for innovation.