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Determining the structure of a protein-DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relative to the DNA within a large transcription complex. We determined the position of the activator on the DNA from data generated using activator proteins that had been conjugated at specific residues with the chemical cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). These analyses were combined with 3-D models of the available structures of portions of the activator protein and B-form DNA to obtain a 3-D picture of the protein relative to the DNA. Finally, the Molsoft program was used to refine the position, revealing the architecture of the protein-DNA within the transcription complex.

A prominent feature of the promoters of Bordetella pertussis fimbrial subunit genes fim2, fim3 and fimX is the presence of a 'C-stretch', a monotonic run of C residues. The C-stretch renders these genes capable of phase variation, through spontaneous variations in its length. For each of these we determined the length of the C-stretch that gave maximal transcriptional activity, and found that the three optimized promoters align perfectly, with identical distances between conserved upstream sequences and the downstream -10 elements and transcriptional start sites. We also demonstrated, for Pfim3, that the conserved sequence corresponds to BvgA binding sites. The more upstream of the two binding sites is predicted to be high affinity, by comparison to a functionally derived consensus BvgA-binding sequence. The other binding site is a fairly poor match to this consensus, with 10 of 14 bp belonging to the C-stretch. Interestingly, the centre of this downstream site of BvgA binding coincides exactly with the centre of the expected typical location of a -35 sequence. However, the lack of a recognizable -35 element (CCCCCC versus TTGACA), and the occupation of this site by BvgA∼P suggest that activation of the fim promoters involves unusual interactions among BvgA, RNA polymerase and promoter DNA.