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M T Record

Publications and source records attributed to M T Record.

105 records · Page 6Linked to original sources
PubMed1977

Measurement of binding constants for protein-DNA interactions by DNA-cellulose chromatography.

We describe the application of DNA-cellulose chromatography to the determination of binding constants for the nonspecific interaction between proteins and DNA. The method involves loading a small DNA-cellulose column to a low binding density and subsequently eluting the protein with buffer at a constant salt concentration. The elution is conveniently followed by monitoring the protein fluorescence of the eluate. From the shape of the elution profile, the binding constant for the interaction can be calculated. Employing columns containing double-stranded calf thymus DNA-cellulose, we have measured binding constants in the range of 10(4) to 10(6) M-1. Extension of this range is possible by varying the amount of DNA on the column. For lac repressor, agreement between our measurements and those of Revzin, A., and von Hippel, P.H. [(1977) Biochemistry 16 (second of five papers in a series in this issue)], who employ an absolute boundary sedimentation method, is good. The column method should be useful for the rapid screening of the effect of a large number of variables on protein-DNA binding constants.

Bacterial Proteins↗
PubMed1977

Nonspecific interaction of lac repressor with DNA: an association reaction driven by counterion release.

We have investigated the nonspecific interaction of lac repressor protein with DNA by a quantitative application of DNA-cellulose chromatography (deHaseth, P.L., et al. (1977), Biochemistry 16 (third of five papers in a series in this issue)). The observed association constant for the interaction, KRD obsd, is a sensitive function of ion concentrations and pH. Application of binding theory to interpret these effects gives the results that 11 +/- 2 monovalent ions are released in the interaction and two groups on repressor must be protonated for repressor to bind to DNA. We argue that much of the ion release results from the displacement of cations from the DNA, and estimate on this basis that 12 +/- 2 phosphates are involved in ionic interactions with the protein. Ion release drives the protonation reaction and the overall repressor-DNA interaction. The major role of low molecular weight ions in the repressor-DNA interaction suggests that ion concentration changes must be considered in discussing mechanisms of control of gene expression.

Bacterial Proteins↗
PubMed1977

Interpretation of monovalent and divalent cation effects on the lac repressor-operator interaction.

We have investigated the effects of mixed Na+: Mg2+ ionic solutions on the stability of the nonspecific lac repressor-DNA complex. The effects of Mg2+ are simply interpreted in terms of its role as a competitor (with repressor) for DNA sites. From these studies, the binding constant of the Mg-DNA complex can be determined as a function of the concentration of Na+. We have used this information to interpret the data of Riggs and collaborators (Riggs, A.D., et al. (1970), J. Mol. Biol. 48, 67-83; 53, 401-417) on the ion dependence of the repressor-operator interaction. We find that there are approximately 70% as many ionic interactions in the repressor-operator complex as in the nonspecific complex. Our best estimate is that 8 +/- 1 ion pairs are formed. We calculate that the release of counterions in the formation of the specific complex contributes approximately 40% of the favorable free energy change in the association reaction under in vivo ionic conditions. Implications of these findings for the control of the lac operon and for the molecular relationship between the specific and nonspecific complexes are considered.

Bacterial Proteins↗
PubMedDate not supplied

Variables affecting the selectivity and efficiency of retention of DNA fragments by E. coli RNA polymerase in the nitrocellulose-filter-binding assay.

In this paper we characterize the effect of varying the solution conditions and filter-binding protocols on the extent and selectivity of DNA retention on nitrocellulose filters by DNA-binding proteins. These effects are illustrated by the binding interaction of Escherichia coli RNA polymerase with lambda and T7 phage DNA restriction fragments. We present procedures which will help enhance the selective retention of some DNA restriction fragments over others. These include increasing the pH and salt concentration, decreasing the enzyme-to-DNA ratio, and including an appropriate washing step. Selective binding is not dependent on the presence of Mg2+. Although we only show data for RNA polymerase-DNA interactions, many of the principles discussed are likely to find practical applications in studying selective DNA-protein binding in general.

Carrier Proteins↗