Nucleic Acids Research, 1992, Vol. 20, No. 4 719-726
© 1992
Articles |
Both fis-dependent and factor-independent upstream activation of the rrnB P1 promoter are face of the helix dependent
Department of Bacteriology, University of Wisconsin 1550 Linden Drive, Madison, Wl 53706, USA
Received December 2, 1991. Revised January 10, 1992. Accepted January 10, 1992.
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.
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