A- and T-tract-mediated intrinsic curvature in native DNA between the binding site of the upstream activator NtrC and the nifLA promoter of Klebsiella pneumoniae facilitates transcription

The nif promoters of Klebsiella pneumoniae must be activated by proteins bo und to upstream sequences which are thought to interact with the sigma(54)- RNA polymerase holoenzyme by DNA looping. NifA is the activator for most of the promoters, and integration host factor (THF) mediates the DNA looping. While NtrC is the activator for the nifLA promoter, no IHF appears to be i nvolved. There are two A tracts and one T tract between the upstream enhanc er and the nifLA promoter. This DNA segment exhibits anomalous electrophore tic mobility, suggesting intrinsic sequence-induced curvature in the DNA. O n the one hand, mutation of the A tracts or T tract individually or togethe r, or deletion of the A tracts and the T tract reduces the anomaly; on the other hand, creation of two additional A tracts enhances the anomaly. Intri nsic curvature in the DNA has been confirmed by circular permutation analys is after cloning the DNA fragment in the vector pBend 2 and also by electro n microscopy. Computer simulation with the DNA base sequence is also sugges tive of intrinsic curvature. A transcriptional fusion with the Escherichia coil lacZ gene of the DNA fragment containing the nifLA promoter and the wi ld-type or the mutated upstream sequences was constructed, and in vivo tran scription in K. pneumoniae and E. coli was monitored. There was indeed very good correlation between the extent of intrinsic curvature of the DNA and transcription from the promoter, suggesting that DNA curvature due to the A tracts and the T tract was necessary for transcription in vivo from the ni fLA promoter of K. pneumoniae.