Conformation of Lac repressor tetramer in solution, bound and unbound to operator DNA.
We tested whether the Steitz et al. [(1974) Proc. Natl. Acad. Sci. U.S.A., 71:593-597] model of lactose repressor (LacR) (14 x 6.0 x 4.5 nm) represented the shape of free or operator-bound LacR in solution. The model predicts a 14 nm length for bound LacR. Direct measurement, using Pt-C shadow width standards, was 9.6 +/- 0.2 nm long. Using the Steitz model, we generated a distribution of measurements and converted them into a distribution of shadow widths using gold ball standards. Direct measurement of LacR produced a narrower shadow width distribution with a larger mean size than the Steitz model predicted. Measurement along two orthogonal axes of negatively stained LacR images generated a size distribution, also converted into a shadow width distribution using the gold ball standards. Since the experimental shadow width distribution exactly matched the shadow width distribution derived theoretically from negatively stained LacR, our negative-stained images are representative of LacR's conformation in solution. Approximately 56% of negatively stained LacR had a V-shaped fold around an axis orthogonal to its length, bringing the DNA binding domains of each dimer adjacent. This open end of the V binds single operator DNA. The other 44% of the LacR tetramer is in the extended form with its DNA binding sites at opposite ends. Although the V-shaped conformation has a closed hinge with the dimers associated along a side, the extended open-hinged state remains important since LacR must bind two distant operator sites for full repression. Our measurements predict the normal presence of both conformations in nearly equal amounts, suggesting that both are equally active in repressing the lac operon.
Ruben
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GC
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755, USA.
Roos
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D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions
D012097:Repressor Proteins; D012996:Solutions; D004247:DNA