1. Distribution of DBDs in the TF Regulatory Network Gene Product Negative regulation with binding site data Positive regulation with binding site data _ Negative regulation without binding site data + Positive regulation without binding site data Homeodomain-like “winged helix” DNA binding domain Lambda repressor-like DNA binding domain C-terminal effector domain of the Bipartite response regulator Putative DNA-binding domain FIS-like IHF like DNA-binding domain Nucleic acid-binding proteins Met repressor-like

2. Information on the regulated gene is available for 113 TFs from RegulonDB. We classified the 113 TFs in to nine functional classes according to what they respond to and what they regulate. Each of these functional classes have one to three proteins (global regulators) that regulate over 50 genes and the other proteins (fine tuners) control fewer than 20 genes Transcription Factors and their regulated genes Transcription Factors and their regulated genes Transcription Factors and their regulated TFs Transcription Factors and their regulated TFs Number of different TF genes (X) regulated by a transcription factor Number of Examples Very few TFs regulate more than one TF The TF network has four major regulatory hubs which are also global regulators in their respective functional class. A relatively complex topology exists in a simple organism

3. Network Growth by Gene Duplication Model-2: Duplication of transcription factor Model-2: Duplication of transcription factor Model-1: Duplication of regulated gene Model-1: Duplication of regulated gene Gene regulatory network in E. coli 400 out of the 1233 (~1/3) interactions have homologous TFs sharing RG or homologous RGs sharing TFs. This suggests that duplication is a major mechanism for network growth 710 out of the 1233 interactions have either TF with a homologue or RG with a homologue but not share RG or TF, suggesting innovation of new edges or duplication followed by loss of regulatory interactions. 123 out of the 1233 interactions have no homologues for TFs and RG suggesting that these interactions were innovations. 32 TFs (out of 110) share regulated genes with a homologue 197 RG (out of 340) share transcription factors with a homologue 197 RG (out of 340) share transcription factors with a homologue Duplication growth models Model-1 Model-2