Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding I: analysis of cro dimerization from nanomolar to micromolar concentrations.
The cro repressor from bacteriophage lambda is an important and classical transcription regulatory protein that binds DNA operator sites as a dimer. Therefore, a complete understanding of gene regulation by cro requires knowledge of the coupled energetics of its protein dimerization and site-specific DNA binding. A method is described by which cro repressor can be labeled in vivo with [(35)S]methionine to a specific activity of 2 x 10(15) cpm/mol. As a prelude to binding studies, the association equilibrium of cro was determined over the range 10(-)(9)-10(-)(3) M using large-zone analytical gel chromatography with radiolabeled repressor. The data are best described by a monomer-dimer stoichiometry with an equilibrium constant of 3.07 (+/-1.08) x 10(6) M(-)(1) total cro monomer. Stokes radii for monomers and dimers were evaluated from the resolved gel partition coefficients. Under the conditions employed in this study (10 mM Bis-Tris, 200 mM KCl, 2.5 mM MgCl(2), 1 mM CaCl(2), 100 microg/mL BSA, pH 7.0, 20 degrees C), self-association of cro to species with assembly states greater than dimers is not observed.
Darling
P J
PJ
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA.
Holt
J M
JM
Ackers
G K
GK
D010582:Bacteriophage lambda / Q000737:chemistry*; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D004734:Energy Metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000821:virology; D008956:Models, Chemical; D008970:Molecular Weight; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013462:Sulfur Radioisotopes / Q000378:metabolism; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D013462:Sulfur Radioisotopes; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins