In vivo DNA loops in araCBAD: size limits and helical repeat

Formation of a DNA loop by AraC proteins bound at the araI and araO2 sites, whose center-to-center distance is 211 base pairs, is necessary for repression of the araBAD promoter, PBAD, of Escherichia coli. To determine the upper and lower size limits of the loop, we constructed PBAD-reporter gene fusion plasmids with various spacings between araI and araO2 and measured their levels of expression. Spacings larger than about 500 base pairs resulted in elimination of detectable repression. No lower limit to spacing was found, suggesting that AraC protein itself possesses significant flexibility and its bending substantially aids formation of small loops. As the spacing between araI and araO2 varied, the activity of PBAD oscillated with an 11.1-base-pair periodicity, implying that the in vivo helical repeat of this DNA is 11.1 base pairs per turn.