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RNA Polymerase II Subunits Exhibit a Broad Distribution of Macromolecular Assembly States in the Interchromatin Space of Cell Nuclei
journal contribution
posted on 2014-01-16, 00:00 authored by Michael A. Tycon, Matthew
K. Daddysman, Christopher J. FeckoNearly
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.
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Keywords
chromatin binding sitesMacromolecular Assembly Statesgene transcription unitsCell NucleiNearlyproteinRNA polymerase II subunitssize extremesRNA Polymerase II Subunits Exhibitchemical interactionsassembly mechanismDrosophila cell nucleiFRAP microscopytranscription complexesRpb 3Broad DistributionInterchromatin SpaceRpb 3 exhibits regionsunengaged subunitsdiffusion dynamics