REGULATION OF THE ESCHERICHIA-COLI NRD OPERON - ROLE OF DNA SUPERCOILING

An in vitro RNA transcription assay was used to investigate the regula tion of the expression of the nrd promoter. Using a linear DNA templat e, we found that Pis protein, which has a positive effect on expressio n of the nrd promoter in an nrd-lacZ fusion in vivo, had a moderate ne gative effect in vitro. However, with a supercoiled DNA template as su bstrate, we found that Fis had a concentration-dependent positive effe ct on nrd transcription in vitro. This positive effect was not present on two templates that had 35- or 37-bp insertions between the Fis bin ding site and the npd promoter. In the absence of Fis protein, a drama tic decrease in transcription was observed in templates with reduced s upercoiling generated by the treatment with wheat germ topoisomerase I . Templates with insertions of 35 bp into an HpaII site at -102 or 37 bp into the MnlI site at -33 bp from the start of transcription failed to exhibit the DNA supercoiling sensitivity of the nrd promoter. Anal ysis of cells containing either of these two nrd-lacZ fusion construct s that has an insertion at the regulatory region by dow cytometry indi cated that these two constructs, unlike the parental construct, were n ot cell cycle regulated.