Structure of recombinant rat UBF by electron image analysis and homology modelling

Abstract

We have studied the structure of recombinant rat UBF (rrUBF), an RNA polymerase I transcription factor, by electron microscopy and image analysis of single particles contrasted with methylamine tungstate. Recombinant rat UBF appeared to be a flat, U-shaped protein with a central region of low density. In the dominant projections, 2-fold mirror symmetry was seen, consistent with the dimerization properties of this molecule, and of dimensions in agreement with the length of DNA that rat UBF protects in footprinting studies. Electron microscopy of various rrUBF-DNA complexes confirmed that our recombinant protein was fully able to bind the 45S rDNA promoter, and that it caused substantial bends in the DNA. Upon extended incubation in a droplet covered by a lipid monolayer at the liquid-air interface, rrUBF formed long filamentous arrays with a railway track appearance. This structure was interpreted to consist of overlapping rrUBF dimers 3.5 nm apart, which value would represent the thickness of the protein. Our results show rrUBF to interact with and bend the promoter DNA into a roughly 10 nm diameter superhelix. Based on all these electron microscopical results, an atomic structure was predicted by homology modelling of the HMG fingers, and connected by energy minimized intervening segments.

Characterization of a Recombinant Murine 18.5-kDa Myelin Basic Protein

“…Moreover, the nature of the repeating motif is unclear, usually resembling a partially closed toroid with a stain pit in the center (cf., 21). A similar protein shape forming extended filaments (although not packed side-by-side) has been reported for another basic protein, the RNA polymerase I transcription factor UBF (upstream binding factor) (92). Interestingly, the rmMBP filaments in Fig.…”

Nucleosome Binding by the Polymerase I Transactivator Upstream Binding Factor Displaces Linker Histone H1

“…Though positive supercoiling could result from overtwisting of the DNA, the simplest explanation is that UBF wraps the DNA in a right-handed direction, completing 360°a pproximately once every ϳ140 bp (two enhancers). Consistent with the latter interpretation, structural studies of rat UBF using high-resolution electron microscopic imaging suggest that the UBF dimer resembles a U in which the free ends are offset to form a right-handed ramp (38). If the DNA follows the contour of the dimer, one would expect the DNA to be wrapped in a right-handed direction.…”

Histone acetylation increases chromatin accessibility

“…This demonstrates that the pore size during metaphase has to be below ~20 nm, which would be the size of the 77 kDa dextrans. As reported previously, smaller transcription factors and chromosomal proteins like UBF, H1, RPA43 and TBP in the size of up to 10 nm diameter [UBF dimer (Neil et al, 1996)] have access to mitotic chromosomes (Chen et al, 2005). Thus, an apparent pore size of 10-20 nm can be estimated for chromatin condensed into mitotic chromosomes.…”