On the interactions of the escherichia coli cyclic-amp receptor and lac repressor with dna
New Haven, Conn., Yale Univ., Diss., 1982
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Tag hinzufügen | Zusammenfassung: | New Haven, Conn., Yale Univ., Diss., 1982 In Escherichia coli, the cyclic AMP receptor (CAP) and the lac repressor modulate transcription of the lac operon. The interactions of these proteins with non-specific DNA and with lac promoter-operator sequences are the topics of this thesis. I have developed a DNA binding assay utilizing gel electrophoresis which permits measurement of the binding stoichiometries, equilibrium constants, and kinetic parameters of protein-DNA interactions. An advantage of this method over existing techniques is that it permits studies of the simultaneous interaction of several proteins with a single DNA molecule. One dimer of CAP binds specifically to each of two lac promoter sites. Uptake of one equivalent of cAMP per dimer is a corequisite for this binding. At 5 (mu)M cAMP the binding constants for the two sites are 8.4 x 10('10) M('-1) and 9.0 x 10('9) M('-1). The dissociation kinetics of the high aaffinity complex are second-order in DNA at 1.6 x 10('-5) M bp and 10 (mu)M cAMP, k(,diss) = 1.5 x 10('-4) sec('-1). Under these conditions association is second-order overall, with k(,a) = 6.6 x 10('6) M('-1) sec('-1). Models for these processes are discussed. In contrast, non-specific DNA binding is 10('5)-fold weaker, is cooperative (w = 10), and is not cAMP dependent. The observation that CAP binds Z as well as B-DNA indicates that a variety of DNA conformations are accommodated in the non-specific binding mode. Lac repressor binds to two sites within the promoter. The operator site is bound (TURN)18-fold more tightly than the secondary site. Each site appears to be bound independently of the other. Pseudo-first order repressor-operator dissociation is temperature sensitive. Below 29(DEGREES)C, (DELTA)E(,a) = 26 kcal mol('-1); above (DELTA)E(,a) = 80 kcal mol('-1). Dissociation of the 2:1 complex is first order in DNA, while dissociation of the 1:1 complex shows rate saturation of high concentrations. These kinetics support the direct transfer model of facilitated protein diffussion between DNA binding sites. |
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| Beschreibung: | 314 S. |