Transcription initiation at certain bacterial promoters can be downregulated by a repressor that binds to a target that overlaps the transcript start site or the −10 element (Gralla and Collado-Vides, 1996). At some of these promoters, the binding of the repressor appears simply to block access of the RNA polymerase holoenzyme (RNAP) to the promoter. However, efficient repression sometimes also requires the binding of another repressor molecule to an auxilliary site that is distant from the transcription start (Muller-Hill, 1998). In some of these cases, the binding of repressor to the auxilliary site improves repression by increasing the local repressor concentration. In other cases, repressor molecules, bound at different sites, combine to form a repression loop, and quite complex nucleo–protein structures form that are essential for repression (e.g. see Choy and Adhya, 1996).
One well-studied example of a complex repressor is the Escherichia coli AraC protein, which functions both as an activator and as a repressor of the transcription of genes necessary for arabinose metabolism. AraC is the founder member of a large family of gene regulatory proteins, the AraC family; the activities of many members of this family are regulated by small ligands (reviewed by Gallegos et al., 1997). AraC is functional as a dimer; in the presence of arabinose, the conformation of AraC is such that the two subunits of the dimer bind to two adjacent target sites resulting in activation of the araBAD promoter. However, in the absence of arabinose, the two subunits of the AraC dimer bind to two well-separated sites, one near the araBAD promoter and the other near the adjacent araC promoter. This results in the formation of a repression loop and the repression of the araC promoter by AraC (reviewed by Schleif, 1996).
In our studies, we have focussed attention on the E. coli MelR protein, which is a member of the AraC family. MelR is essential for induction of the melAB operon that is responsible for melibiose metabolism. MelR is encoded by the melR gene that is located upstream from the melAB promoter (Webster et al., 1987). In previous work, we characterized the melR promoter; it is divergent from the melAB promoter, with the melR and melAB transcript starts separated by 237 bp (Webster et al., 1988). Expression from the melR promoter is completely dependent on the cyclic AMP receptor protein (CRP), which binds to a DNA site centered at position −41.5 upstream from the melR transcript start point (Webster et al., 1988). MelR activity is regulated by melibiose, which is required for activation of transcription initiation at the melAB promoter (Webster et al., 1989). MelR binds to 18 bp targets located at the melAB promoter (Caswell et al., 1992; Williams et al., 1994). In the accompanying paper (Belyaeva et al., 2000), we showed that, in the absence of melibiose, MelR binds to three 18 bp target sites (Site 1, Site 1′ and Site 2) located just upstream of the melAB transcription start site. Melibiose induces the occupation of a fourth site (Site 2′) which is essential for transcription initiation at the melAB promoter (Fig. 1). During this work, and in a previous study (Gostick et al., 1998), it was noted that MelR could also bind to a fifth site (Site R) that overlaps the transcription start point and −10 hexamer of the melR promoter (Fig. 1). Our in vitro studies, showed that MelR can bind to this site in both the presence and absence of melibiose, that this site accommodates just one MelR subunit, and that binding is tightened by CRP (Belyaeva et al., 2000; T. A. Belyaeva, unpublished data). In this study, we have investigated whether the binding of MelR to this site affects expression from the melR promoter. We show that the melR promoter is repressed by MelR and that this autoregulation requires MelR binding to Site R. In the absence of melibiose, greater repression occurs in the presence of MelR binding Site 2, located 177 bp upstream. We propose that, for optimal repression, MelR forms a loop between Site R and Site 2, and that this loop is broken in the presence of melibiose when the melAB promoter is activated.
Figure 1. Schematic diagram of the KK81 and KK101 fragments.The figure illustrates the KK81 fragment carrying the divergent melAB and melR promoters, and the KK101 fragment carrying just the melR promoter. Horizontal arrows indicate the transcription start sites. DNA sequences are numbered with respect to the melR transcript start as +1. Thus, the KK81 and KK101 fragments are bounded by EcoRI sites at position +76 downstream of the melR promoter, and HindIII sites at positions −272 and −74, respectively, upstream. The locations of the different DNA sites for MelR are indicated by triangles: each triangle indicates an 18 bp sequence and the position of the centre of each site is numbered (see Belyaeva et al., 2000). The filled triangles indicate Site 1 and Site 2, the grey triangles denote Site 1′ and Site R, and the open triangle denotes Site 2′ that is filled by MelR only in the presence of melibiose. The shaded boxes denote 22 bp DNA sites for CRP: one site centered at position −41.5 is responsible for the activation of the melR promoter, while the other, centered at position −155.5 is involved in activation of the melAB promoter. The complete base sequence of the KK81 fragment is given in Fig. 1B of the accompanying paper (Belyaeva et al., 2000).
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