Sequence characteristics required for cooperative binding and efficient in vivo titration of the replication...

Plasmids carrying the mioC promoter region, which contains two DnaA boxes, R5 and R6 with one misfit to the consensus TT(A)/(T)TNCACA, are as efficient in in vivo titration of the DnaA protein as plasmids carrying a replication-inactivated oriC region with its eight DnaA boxes. Three additional DnaA boxes around the promoter proximal R5 DnaA box were identified and shown by mutational analysis to be necessary for the cooperative binding of DnaA required for titration. These four DnaA boxes are located in the same orientation and with a spacing of two or three base-pairs. The cooperative binding was eliminated by insertion of half a helical turn between any of the DnaA boxes. Titration strongly depends on the presence and orientation of the promoter distal R6 DnaA box located 104 bp upstream of the R5 box as well as neighbouring sequences downstream of R6. Titration depends on the integrity of a 43 bp region containing the R5 DnaA box, while repression of mioC transcription by DnaA, which is dependent on the R5 DnaA box, was independent of the two DnaA boxes downstream of R5. Repression was also independent of the spacing between the two upstream DnaA boxes and the promoter as long as a DnaA box was located less than 20 bp upstream of the -35 sequence. Thus, the architectural requirements for titration and for repression of transcription are different. A new set of rules for identifying efficiently titrating DnaA box regions was formulated and used to analyse sequences for which good titration data are available.