Functional and physical characterization of transcription initiation complexes in the bacteriophage lambda OR region

We have used transcriptional activity assays and DNase I footprinting techniques to examine in vitro the binding of Escherichia coli RNA polymerase and lambda repressor protein to the bacteriophage lambda rightward promoter-operator region. For the lambda PR promoter, the activity and physical binding results determined at several repressor concentrations correlated very well. Good agreement was also found for repression of PRM, which occurred at higher repressor concentrations; however, our results indicate that at low repressor concentrations, RNA polymerase can physically occupy PRM in a transcriptionally inactive form. These inactive complexes formed with a binding constant similar to that previously measured for "closed complexes" at PRM. A kinetic study of PR open complex formation on an OR2-template in the presence of lambda repressor showed that decreased initiation frequency from this promoter was due largely to a decrease in KB. The kinetically determined inhibition constant for repressor (Ki = 4 nM) was similar to the dissociation constant (Kd approximately 2 nM) determined from the footprinting studies.