RESUMO
Transcriptional activation by the E.coli NtrC protein can occur via DNA looping between a DNA-bound activator and the target sigma(54) RNA polymerase. NtrC forms an octamer on DNA that is capable of binding two DNA molecules. Its ATPase activity is required for open complex formation. Geometric requirements for activation were assessed using a library of DNA bending sequences created by random ligation of A-tract oligonucleotides, as well as several designed sequences. Thirty random or designed sequences with a variety of DNA lengths and bending geometries were cloned in plasmids, and the library was used to replace the spacer between the NtrC binding sites and the core glnAp2 promoter. The activity of each promoter construct under nitrogen limitation was determined in vivo, in a lambda phage lacZ reporter system integrated as a single-copy lysogen to avoid titrating NtrC or polymerase. A wide variety of bending geometries was found to support a similar level of transcriptional activation ( approximately 3-4-fold). Computer modeling of the DNA trajectories suggests that the most inactive promoters have short spacer DNA and the NtrC sites on the opposite side of the helix as the wild-type sites; otherwise, the loop can form effectively. Flexibility and multivalency of the NtrC-Esigma(54) interaction apparently provides substantial independence from DNA stiffness constraints, and in general activation requires less efficient looping than repression. However, none of the random templates were as active as wild-type promoter. Subsidiary activator binding sites in the wild-type were found to be required for full activity, but, surprisingly, these sites could not be functionally replaced by strong binding sites. This suggests that one or more protomers in the NtrC octamer must form and then release contacts with DNA in order to complete the ATPase cycle and act as an AAA(+) activator of the Esigma(54). This dynamic DNA wrapping around the NtrC octamer is proposed to be necessary for efficient activation, and the wrapping may also reduce adventitious activation of other promoters.
