Transcription Activation Parameters at ara pBAD

Abstract

We studied the formation of open complexes of RNA polymerase and promoter DNA as activated by the AraC protein at the Escherichia coli araBAD promoter pBAD and by the cyclic AMP receptor protein at the galKTE promoter P1. The DNA migration retardation assay was demonstrated to be suitable for the detection and quantitation of open complexes by the correspondence in the properties of open complexes in solution and retarded complexes observed in gels. These included, on the ara promoter, heparin resistance, lifetime, DNAseI footprinting, exonuclease III footprinting, permanganate footprinting and disappearance upon transcription, and on the gal promoter, the correspondence between the kinetic parameters Kd and k2 obtained with established techniques and those obtained with the migration retardation assay On the PBAD promoter we obtained kinetic parameters of Kd = 0.3 nM and k2 = 1 minute-1. The unusually tight binding of polymerase in the presence of AraC suggests that AraC binds polymerase tightly. © 1996 Academic Press Limited.