Interactions protéines — acides nucléiques

To study the binding of proteins to DNA, it was found useful to develop a general method of interpretation of binding isotherms, which appears as an extension of previous calculations made by Latt and Sober. It is assumed that electrostatic site‐binding only occurs between adjacent phosphate groups and basic residues of the protein. For each molecule of protein a number a of electrostatic bonds are made and m sequences of a sites are available on the DNA. When all the sites are available ( ma equal to the total number of nucleotides) isotherms have to be calculated by means of a computer. If K is the intrinsic binding constant, A o the concentration of the free protein and v̄ the average number of mole bound to DNA, a plot of 1/v̄ versus 1/ KA o is found to be practically linear in a large range of KA o values. The easiest way to interprete the linear part of the isotherms quantitatively is to recalculate it by using the Lifson's method of sequence generating function. One gets the simple equation 1/ r = a +γ+ (1/ KA o ) where r is the amount of bound protein per nucleotide and γ a parameter determined graphically which depends only on a , and on range of KA o . Values of a and K are thus readily obtained. The number of sites involved in the binding may be smaller than the total number of phosphate groups in the DNA, either by steric hindrance of the protein and hence masking of a certain number of phosphate groups or by the existence of specific sequences of nucleotides. In the first case, if b is the number of sites covered by the protein, equation, of the linear part of the isotherms is now 1/ r = b +γ+ (1/ KA o ) and only b and K can readily be measured. It is however possible to determine a by means of competition experiment between proteins and counter‐ions for which all the sites are assumed to be available. If K′ is now the apparent association constant, the change of K′ with ionic strength is represented by log K′ = a log K o + a log [ k 1/(1 + k 1 c 1 )] where k 1 and c 1 are respectively the association constant with DNA and the concentration of counter‐ions and K o a parameter taking into account the non coulombian binding energy between basic groups of the proteins and phosphate groups. The plot of log K′ versus log [ k 1 /(1 + k 1 c 1 )] enables the determination of K o . In the second case m and K are readily obtained from isotherms by using the classical theory of multiple equilibria and a is determined according to the method previously described. Such a method was tested on oligolysine—polyribonucleotide interactions where calculated values of a are in agreement with those expected. The method was also applied to the binding of repressor to DNA for which only three phosphate groups seem to be involved in the process. Electrostatic energy, which was predominant in the former case, is only a small part of the total energy of binding in the latter.