POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
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<strong>BLAST</strong> X Poster #42<br />
INTERACTION OF THE TRANSCRIPTIONAL REGULATORY COMPLEX, FlhDC, WITH ITS<br />
TARGET DNA<br />
Yi-Ying Lee, and Philip Matsumura<br />
Department <strong>of</strong> Microbiology and Immunology, College <strong>of</strong> Medicine, <strong>University</strong> <strong>of</strong> Illinois at<br />
Chicago, 835 S. Wolcott Ave., M/ C 790, Chicago, Illinois 60612-7344<br />
The bacterial flagellum is the structure that allows bacteria to move and respond to<br />
nutritional and chemical signals in their environment. It is a complex suborganelle and the<br />
transcriptional regulation <strong>of</strong> the 40 plus structural genes is organized in a highly regulated<br />
cascade. At the top <strong>of</strong> the hierarchy is the master operon which codes for FlhD and FlhC. These<br />
two positive transcriptional regulators form a unique heteroheximeric complex which binds<br />
upstream <strong>of</strong> the -35 region and requires sigma 70 for transcription. This complex has an<br />
unusually large ‘footprint’ <strong>of</strong> 48 base pair and bends the DNA 110 degrees. We have proposed<br />
that the DNA bind on the circumference <strong>of</strong> this toroid shaped FlhDC complex. Although we have<br />
determined the sequence 3 footprints on FlhDC regulated promoters, it is not possible to<br />
determine a consensus binding site in these 3 sequences. In this study, we have determined<br />
which bases are important for DNA binding and activity for FlhDC regulated promoter activity.<br />
First, we have divided the FlhDC footprint in the fliA promoter into five segments and found that<br />
two <strong>of</strong> the segments or 40% <strong>of</strong> the footprint were not required for binding. The remaining 30<br />
base pairs were divided into 3-5 base segments and randomly mutagenized and screened for<br />
the ability to bind and activate the fliA promoter. Analysis <strong>of</strong> these data suggests a consensus <strong>of</strong><br />
12 A, 15 A, 34 T, 36 A, 37 T, 44 A, 45 T in FlhD4C2 footprint fragment were important for activity. Five <strong>of</strong><br />
these bases demonstrated high specificity. Finally, this consensus was tested and found to be<br />
important in other FlhDC regulated promoter regions.<br />
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