Abstract
The affinity of synthetic P22 operators for P22 repressor varies with the base sequence at the operator's center. At 100 mM KCl, the affinity of these operators for P22 repressor varies over a 10-fold range. Dimethylsulfate protection experiments indicate that the central bases of the P22 operator are not contacted by the repressor. The KD for the complex of P22 repressor with an operator bearing central T-A bases (9T) increases less than 2-fold between 50 and 200 mM KCl, whereas the KD for the complex of repressor with an operator bearing central C-G bases (9C) increases 10-fold in the same salt range. The DNase I cleavage patterns of both bound and unbound P22 operators also vary with central base sequence. The DNase I pattern of the repressor-9C operator complex changes markedly with salt concentration, whereas that of the 9T operator-repressor complex does not. These changes in nuclease digestion pattern thereby mirror the salt-dependent changes in the P22 operator's affinity for repressor. P22 repressor protects the central base pair of the 9T operator from cleavage by the intercalative cleavage reagent Cu(I)-phenanthroline, while repressor does not protect the central bases of the 9C operator. Together these data indicate that central base pairs affect P22 operator strength by altering the structure of the unbound operator and the repressor-operator complex.
Highlights
ATTTA AT GC AT TAAATS9C0 ATTTAAGACGTCTTAAAT swap experiment shows that when P22amino acid sequences are grafted onto a protein that is sensitive to the sequences at the center of the operator, the 434 repressor, the resulting hybrid protein has the same order of preferencefor the binding sites in P22 OR as doeswild-type P22 repressor (Wharton and Ptashne 985).This suggeststhat P22 repres-
The affinity of synthetic P22 operators for P22 re- ators iscritical for the growth life cycle choices of the bactepressor varies with the base sequence at the operatorri’osphage
The Kr, for the repressor-9T operator complex is relatively independent of KC1 concentration, whereas that for 9C operator-repressor complex is more salt-sensitive (Fig. 3)
Summary
S9C0 ATTTAAGACGTCTTAAAT swap experiment shows that when P22amino acid sequences are grafted onto a protein that is sensitive to the sequences at the center of the operator, the 434 repressor, the resulting hybrid protein has the same order of preferencefor the binding sites in P22 OR as doeswild-type P22 repressor (Wharton and Ptashne 985).This suggeststhat P22 repres-. EXPERIMENTALPROCEDURES of binding protein; results of experiments performed with a 3-fold higher protein concentration were not qualitatively or quantitatively. The sequences of the binding sites used are given in Fig. 1.The sequences of the resulting plasmids were confirmed by dideoxy methods These DNA molecules were cleaved a t the EcoRI site and 3'-end-labeled by repairing the recessed ends with Klenow and [w3'P]dATP or 5'-end-labeled with [y3'P]ATP andpolynucleotide kinase. These -2700-base pair linear DNAs were used directly in filter binding studies.
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