POSTERS - BLAST X - University of Utah

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