The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro

Abstract

The lactose promoter-operator region of Escherichia coli contains two binding sites for cyclic AMP receptor protein (CAP), two for the lactose repressor, and two for RNA polymerase. The high density of binding sites makes cooperative interactions between these proteins likely. In this study, we used the gel electrophoresis mobility shift assay and binding partition analysis techniques to determine whether the secondary CAP site influences the binding of CAP to the principal CAP site in the lactose promoter when both are present on a linear DNA molecule. Such an effect could occur through the formation of a bridged DNA-CAP-DNA structure, through the interaction of CAP molecules bound to each of the sites, or through allosteric effects caused by CAP-mediated DNA bending. We found, however, that the interaction of CAP with these sites was not cooperative, indicating that CAP sites 1 and 2 bind CAP in an independent manner.

Cation binding linked to a sequence-specific CAP–DNA interaction

“…The site designated CAP1 is the genetically-defined locus of gene activation. For the range of binding conditions used in this study, K obs site 1 >20 K obs site 2 [6,28,36]. Accordingly, CAP is predominantly bound at site 1 in the 1:1 complexes considered here.…”

Effects of anions on the binding of the cAMP receptor protein to the lactose promoter

“…2A shows a typical mobility-shift experiment, allowing the determination of free and CAP-bound DNA concentrations as a function of [CAP]. We have previously shown that the protein-DNA complex designated C1 consists of CAP bound to CAP site 1 (Hudson and Fried, 1991). For binding in buffer A supplemented with 25 mM potassium salts, Kobs ranged from 1.2 ( 2 0.3) X loio M-' (potassium acetate) to 2.3 (2 0.6) X lo9 M-' (potassium glutamate).…”

“…Binding was carried out at 21 2 1°C. Complex C l contains one CAP dimer/DNA fragment bound to CAP site 1 (Fried, 1982;Hudson and Fried, 1991 on going from 25 mM to 100 mM salt. This order is similar to that reported for the effect of anion substitution on the binding of Zac repressor to operator DNA (Barkley et al, 1981), and resembles orders found for the salting out of RNase A (von Hippel and Wong, 1965), the melting of duplex DNA (Hamaguchi and Geiduschek, 1962), and the thermal precipitation of duplex DNA in the presence of divalent cations (Knoll et al, 1988).…”

“…cAMP activation of the lactose promoter is lac site 1 (Beckwith et al, 1972). In vitro studies with lac promoter DNA have shown that as CAP concentration is increased, the first stable complex to form contains one dimer of CAP/DNA molecule, with predominant occupancy of lac site 1 ; further addition of CAP results in the formation of a 2 : 1 complex with lac sites 1 and 2 both occupied (Fried and Crothers, 1984;Hudson and Fried, 1991). Once bound, CAP exerts several effects on the proteins that occupy adjacent sites on the DNA.…”

Bioinformatics Resources for the Study of Gene Regulation in Bacteria

“…TFs of the MerR family activate transcription binding at a palindrome located between the Ϫ35 and Ϫ10 elements of the promoter (71,93). Other regulators activate transcription through auxiliary binding sites located between Ϫ10 and ϩ20 (38). A striking, well-documented case is activation from ϩ1848 by IHF, a TF known to bend the DNA to favor transcription (1).…”