Differential regulation of multiple overlapping promoters in flagellar class II operons in Escherichia coli

Abstract

The Escherichia coli flagellar operons are divided into three categories: classes I, II and III. Expression of class II depends on expression of class I. One of the class II gene products, the FIIA protein, is an alternative sigma factor (sigma 28) required for transcription of the class III operons. In this study, we have characterized, in vitro, a role of sigma 28 in the regulation of the class II operons. Among the three class II operons examined, the fliA and fliL operons, but not the flhB operon, could be transcribed by both sigma 70 RNA polymerase holoenzyme with FihD/C (E sigma 70-FlhD/C) and sigma 28 RNA polymerase holoenzyme (E sigma 28). The flhB operon could only be transcribed by E sigma 70-FlhD/C under the conditions used. Both the fliA and fliL operons contained two overlapping promoters oriented in tandem. The transcription of fliA directed by E sigma 28 could outcompete that by E sigma 70-FlhD/C, indicating a positive autoregulation. However, E sigma 28 could not displace E sigma 70-FlhD/C bound to the fliL promoter. The sigma 28-mediated positive regulation of the class II operons involved a mechanism in which sigma 28 competed with sigma 70 for core RNA polymerase. In addition, recruitment of core RNA polymerase from the sigma 70 -10 site to the sigma 28 -10 was facilitated by formation of E sigma 70-FlhD/C pre-initiation complex. Taken together, the three class II promoters investigated are different in terms of their regulation by sigma 28. We propose that class II operons may be further divided into different subcategories.