title: RNA polymerase II subunits exhibit a broad distribution of macromolecular assembly states in the interchromatin space of cell nuclei.
abstract: Nearly all cellular processes are enacted by multi-subunit protein complexes, yet the assembly mechanism of most complexes is not well understood. The anthropomorphism "protein recruitment" that is used to describe the concerted binding of proteins to accomplish a specific function conceals significant uncertainty about the underlying physical phenomena and chemical interactions governing the formation of macromolecular complexes. We address this deficiency by investigating the diffusion dynamics of two RNA polymerase II subunits, Rpb3 and Rpb9, in regions of live Drosophila cell nuclei that are devoid of chromatin binding sites. Using FRAP microscopy, we demonstrate that both unengaged subunits are incorporated into a broad distribution of complexes, with sizes ranging from free (unincorporated) proteins to those that have been predicted for fully assembled gene transcription units. In live cells, Rpb3 exhibits regions of stability at both size extremes connected by a continuous distribution of complexes. Corresponding measurements on cellular extracts reveal a distribution that retains peaks at the extremes but not in between, suggesting that partially assembled complexes are less stable. We propose that the broad distribution of macromolecular species allows for mechanistic flexibility in the assembly of transcription complexes.
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-3290, United States.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Tycon
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Daddysman
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Fecko
mesh_terms: D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D004058:Diffusion; D004330:Drosophila / Q000254:growth & development; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007814:Larva / Q000201:enzymology / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012469:Salivary Glands / Q000201:enzymology / Q000378:metabolism
chemical_list: D002843:Chromatin; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012319:RNA Polymerase II