We cloned a consensus DNA site for the Escherichia coli FNR protein at different locations upstream of the E. coli melR promoter. FNR can activate transcription initiation at the melR promoter when the FNR binding site is centered around 41, 61, 71, 82, and 92 bp upstream from the transcription start. The SF73 positive control amino acid substitution in FNR interfered with transcription activation by FNR in each case. In contrast, the GA85 positive control substitution reduced activation only at the promoter, where the FNR binding site is 41 bp upstream of the transcript start. The SF73 substitution appears to identify an activating region of FNR that is important for transcription activation at promoters that differ in architecture. Experiments with oriented heterodimers showed that this activating region is functional in the upstream subunit of the FNR dimer at the promoter where FNR binds around 41 bp from the transcript start and in the downstream subunit at the promoters where FNR binds farther upstream.The Escherichia coli FNR and CRP proteins are both global activators of transcription initiation, interacting at a large number of promoters, their activity being triggered by oxygen and glucose starvation, respectively. FNR and CRP possess related primary sequences, and it is presumed that they have homologous structures and evolved from a common origin (for reviews, see references 8 and 10).Target sites for both FNR and CRP span 22 bp, accommodating dimers of both activators. A striking feature of CRPdependent promoters is that there is considerable variation in the location of the CRP binding site from one promoter to another. In contrast, at most naturally occurring FNR-dependent promoters, the 22-bp DNA site for FNR is centered near Ϫ41 (8, 10). Studies in which the same consensus CRP-binding site was cloned at different distances upstream of the same promoter sequence showed that bound CRP could activate transcription when the center of the CRP dimer was located around Ϫ41, Ϫ61, Ϫ71, Ϫ81, or Ϫ91 bp upstream from the start site (7,11). In a parallel study, Bell and coworkers (2) cloned the same DNA site for FNR at different distances upstream of the same core promoter sequence (covering the Ϫ35 region, the Ϫ10 region, and the transcript start). Experiments with the resulting series of promoters showed that FNR was an efficient transcription activator when centered near Ϫ41 but functioned poorly when centered near Ϫ61 and failed to activate transcription when bound farther upstream (2). We reexamined this observation in this work: we found that alteration of the Ϫ35 region of the core promoter sequence permits the construction of a series of semisynthetic FNR-triggered promoters that are active when the DNA site for FNR is centered near Ϫ41, Ϫ61, Ϫ71, Ϫ82, and Ϫ92.By analogy with CRP, it is probable that FNR activates transcription at target promoters by making direct contact with RNA polymerase (4,8,13). The locations of these contact sites in FNR have been mapped by positive control substitutions that ...

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A library of random mutations in the Escherichia coli fnr gene has been screened to identify positive control mutants of FNR that are defective in transcription activation at Class I promoters. Single amino acid substitutions at D43, R72, S73, T118, M120, F181, F186, S187 and F191 identify a surface of FNR that is essential for activation which, presumably, makes contact with the C-terminal domain of the RNA polymerase alpha subunit. This surface is larger than the corresponding activating surface of the related transcription activator, CRP. To identify the contact surface in the C-terminal domain of the RNA polymerase alpha subunit, a library of mutations in the rpoA gene was screened for alpha mutants that interfered with transcription activation at Class I FNR-dependent promoters. Activation was reduced by deletions of the alpha C-terminal domain, by substitutions known to affect DNA binding by alpha, by substitutions at E261 and by substitutions at L300, E302, D305, A308, G315 and R317 that appear to identify contact surfaces of alpha that are likely to make contact with FNR at Class I promoters. Again, this surface differs from the surface used by CRP at Class I CRP-dependent promoters.

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