Prediction of regulatory proteins in the E.coli genome. The method is divided into two stages. In the first stage, a set of putative transcription factors is generated. This results from searches in the SwissProt database (keywords and regular expressions were used) and from scanning all 4283 ORFs of the E.coli genome using Dodd and Egan, PROSITE patterns and Gibbs sampler algorithms. In addition, a literature search was performed for evidence concerning new transcription factors. We filtered proteins that are not transcription regulators (a transcription factor has a DNA-binding functional motif). In the second part, a sequence comparison was performed using Blast 2.0 to detect additional proteins. Additionally, annotations of the E.coli genome were used together with the collection in RegulonDB (3,32). The DNA-binding protein structures used in the Gibbs sampler calibration were: ArgR (58), BirA (59,60), Crp (61), Fis (62), FruR (63), LacI (64), LexA (65), NarL (66), OmpR (67), PurR (68), TetR (69,70) and TrpR (71).

HTH distribution in the complete set of E.coli proteins. The distribution of the relative HTH location is shown for different subsets of the collection. On the x-axis, 0% represents the N-terminus and 100% the C-terminus end of the protein. The y-axis shows the frequency of regulators. A total of 234 transcriptional factors bearing an HTH motif were taken into account for this analysis. Closed triangles, repressors; closed circles, activators; open circles, dual proteins (activator and repressor); open triangles, those with unknown function.

Hypothetical alternative evolutionary pathways for the emergence of current transcription regulatory families. A DBD with a length of ~60–100 amino acid residues has at least two pathways along which to evolve into protein families. In the first group, a second or third domain (with length relatively conserved) was added to the DBD, to its N- or C-terminus. This fusion produced as a consequence the divergence of several members of the family as a whole unit. In the second heterogeneous group, several domains were added to the N- or C-terminus of the DBD. As a consequence, several sub-groups depending on the size, physiological function and regulatory role should be identified in the same family.