7582 Nucleic Acids Research, 2007, Vol. 35, No. 22 Figure 1. Distribution of number of genes contained in EcoCyc transcription units (TUs). For example, 110 TUs are four genes in length. Figure 2. Histogram of the number of transcription factors (TFs) that regulate a given number of transcription units (TUs). For example, there are 13 TFs where each of those TFs individually regulates three TUs. Put another way, this ﬁgure shows the distribution of E. coli regulon sizes. This ﬁgure diﬀers from the next ﬁgure in various ways; for example, this ﬁgure shows how many occurrences there are where three diﬀerent TUs each contain promoter-proximal binding sites for the same TF (13), whereas the next ﬁgure shows how many occurrences there are of a given TU containing promoter-proximal binding sites for three diﬀerent TFs. The latter cases are also called ‘in-coming’ interactions, whose distribution is known to follow the power law. promoters, of which 1625 have experimental evidence. A total of 706 TUs contain at least one deﬁned TF-binding site. Most EcoCyc TUs lack binding sites because most TUs were predicted computationally, and their TF-binding sites were not predicted. More than one- third of those TUs that contain identiﬁed binding sites contain only one binding site; however, 19 TUs contain more than 10 TF-binding sites each. EcoCyc contains 171 TFs with literature-derived transcriptional regulatory relationships involving one or more TF-binding sites. TFs are highly variable in the number of TUs that they regulate; 44 factors regulate one TU each, whereas two factors each regulate more than 100 TUs. Figure 2 provides the distribution of TF regulon size and identiﬁes the global regulators of E. coli. Figure 3 shows the converse relationship, namely, Figure 3. Histogram of the number of TUs that are jointly regulated by a given number of TFs. For example, 31 TUs are regulated by four TFs, meaning that each of those 31 TUs contains promoter-proximal binding sites for four diﬀerent TFs. the distribution of the number of TFs that regulate each TU. A total of 156 TUs are regulated by three or more TFs. EcoCyc contains DB objects that deﬁne interactions between TFs and 48 small-molecule ligands that regulate TF activity. Table 4 shows the TF ligands that are deﬁned in EcoCyc. A total of 109 EcoCyc TFs lack deﬁned ligands. In many cases, this is because the TFs interact with sensor kinases that themselves interact with the ligands, but we expect that ligands remain to be curated or elucidated for many of these TFs. The interaction between each TF with each binding site is described by a separate interaction object within EcoCyc. These interaction objects also describe which sigma factor recognizes each promoter. Figure 4 shows the number of promoters controlled by each E. coli sigma factor, as reported in the literature. What fraction of the E. coli transcriptional network has been experimentally elucidated to date? Of the roughly 330 E. coli genes that encode TFs, at least one binding site has been experimentally determined for approximately half of them. It is likely that more binding sites for these TFs remain to be determined. A total of 23.0% of TUs have at least one experimentally determined binding site, and 23.8% of TUs have experimental evidence for the extent of the TU. Although our knowl- edge of the regulatory network is clearly incomplete, experimentalists have probably determined in the order of 25–30% of the regulatory interactions within the transcriptional regulatory network. The Pathway Tools software (5) provides EcoCyc with several mechanisms for visualizing regulatory informa- tion. A new visualization of the full transcriptional regulatory network in EcoCyc, called the Regulatory Overview, is shown in Figure 5. The user can interactively explore regulatory relationships on this diagram through several diﬀerent queries, such as displaying arrows linking gene G to all the genes that G regulates, or to all the genes that regulate G. In addition, the user can selectively highlight within a diagram of the full metabolic map of E. coli (see http://biocyc.org/ECOLI/NEW- IMAGE?type=OVERVIEW) those enzymes that are transcriptionally regulated by a speciﬁed transcription factor. These operations are currently available only