Use of difference boundary sedimentation velocity to investigate nonspecific protein-nucleic acid interactions.
The difference boundary sedimentation velocity technique of Schachman and co-workers is demonstrated to be applicalbe to the measurement of binding constants (Kobsd) in the range 10(2)-10(5) M(-1) for the nonspecific interactions of proteins with DNA. The difference technique can reproducibly detect a 2% change in the sedimentation coefficient of the DNA upon binding ligands, corresponding to average extents of association as low as 10 molecules of protein (in the cases of Escherichia coli lac repressor and E. coli RNA polymerase) per molecule of bacteriophage T7 DNA. At these low binding densities, it is plausible to assume that the primary effect of ligand binding is on the buoyant mass of the complex and not on the frictional coefficient of the flexible DNA coil. Binding constants calculated by using this assumption agree well with literature values for the nonspecific interactions of RNase and lac repressor proteins with double-stranded DNA. Advantages of the method are that it is relatively rapid, requires the optical detection of the DNA only, and can be performed on small amounts of sample. The method appears useful for surveying (to an accuracy of +/-50% in Kobsd or +/-10% in log Kobsd) the effects of solution variables on Kobsd of protein-DNA interactions. Applications of the method to the nonspecific interactions of RNA polymerase core and holoenzymes with T7 DNA are discussed.
D000818:Animals; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases; D004720:Endonucleases; D004926:Escherichia coli / Q000378:metabolism; D008024:Ligands; D008433:Mathematics; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D012097:Repressor Proteins; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases; D013604:T-Phages; D014157:Transcription Factors; D014461:Ultracentrifugation / Q000379:methods
D004279:DNA, Viral; D008024:Ligands; D012097:Repressor Proteins; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; D004720:Endonucleases; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic