Structure of recombinant rat UBF by electron image analysis and homology modelling.
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.
Neil
K J
KJ
Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.
Ridsdale
R A
RA
Rutherford
B
B
Taylor
L
L
Larson
D E
DE
Glibetic
M
M
Rothblum
L I
LI
Harauz
G
G
D000818:Animals; D003198:Computer Simulation; D003460:Crystallization; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D008055:Lipids / Q000737:chemistry; D008854:Microscopy, Electron; D008958:Models, Molecular; D035463:Pol1 Transcription Initiation Complex Proteins; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*
D004268:DNA-Binding Proteins; D008055:Lipids; D035463:Pol1 Transcription Initiation Complex Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C063844:transcription factor UBF; D004247:DNA