Both fis-dependent and factor-independent upstream activation of the rrnB P1 promoter are face of the helix dependent

Abstract

Transcription from the Escherichia coli rrnB P1 promoter is increased by a cis-acting sequence which extends upstream of the - 35 hexamer to about -150 with respect to the transcription initiation site, the Upstream Activation Region (UAR). Activation by the UAR involves two components: (1) a trans-acting protein, Fis, which binds to three sites in the UAR between -60 and -150, and (2) the UAR sequences themselves which affect RNA polymerase (RNAP) activity Independent of other proteins. We refer to the latter as Factor-Independent Activation (FIA). In addition to its interactions with the -10 and -35 hexamers typical of E. coll promoters, RNAP makes contacts to the - 53 region of rrnB P1, which may be related to the FIA effect. We constructed a series of insertion mutants containing Integral and non-integral numbers of helical turns at position -46, between the Fis binding sites and the - 35 region, and the resulting promoter activities were measured in vitro and In vivo. The data suggest that both Fis-dependent and factor-independent activation are face of the helix dependent: the Fis binding site and the sequences responsible for factor-independent activation must be correctly oriented relative to RNA polymerase in order to activate transcription. These results, in conjunction with other evidence, support a model for the Involvement of direct Fis-RNAP interactions in upstream activation. We also demonstrate that RNAP interacts with the - 53 region of the rrnB P1 UAR even when these sequences are displaced upstream of the RNAP binding site, and that these interactions correlate with factor-independent activation. © 1992 IRL Press at Oxford University Press.