Evolutionary Dynamics of Prokaryotic Transcriptional Regulatory Networks M. Madan Babu 1,2 * , Sarah A. Teichmann 2 and L. Aravind 1 * 1 National Center for Biotech- nology Information, National Institutes of Health, MD 20894 USA 2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB22QH, UK The structure of complex transcriptional regulatory networks has been studied extensively in certain model organisms. However, the evolutionary dynamics of these networks across organisms, which would reveal important principles of adaptive regulatory changes, are poorly under- stood. We use the known transcriptional regulatory network of Escherichia coli to analyse the conservation patterns of this network across 175 prokaryotic genomes, and predict components of the regulatory networks for these organisms. We observe that transcription factors are typically less conserved than their target genes and evolve independently of them, with different organisms evolving distinct repertoires of transcription factors responding to specific signals. We show that prokaryotic transcriptional regulatory networks have evolved principally through widespread tinkering of transcriptional interactions at the local level by embedding orthologous genes in different types of regulatory motifs. Different transcription factors have emerged independently as dominant regulatory hubs in various organisms, suggesting that they have convergently acquired similar network structures approximating a scale-free topology. We note that organisms with similar lifestyles across a wide phylogenetic range tend to conserve equivalent interactions and network motifs. Thus, organism-specific optimal network designs appear to have evolved due to selection for specific transcription factors and transcriptional interactions, allowing responses to prevalent environmental stimuli. The methods for biological network analysis introduced here can be applied generally to study other networks, and these predictions can be used to guide specific experiments. q 2006 Published by Elsevier Ltd. Keywords: transcriptional regulatory network; evolution; network; network motif; regulation *Corresponding authors Introduction Of the several steps at which the flow of information from a gene to its protein product is controlled, regulation at the transcriptional level is a fundamental mechanism observed in all organisms. This form of regulation is typically mediated by a DNA-binding protein (transcription factor) that binds to target sites in the genome and, either singly or in combination with other factors, regulates the expression of one or more target genes. The sum total of such transcriptional interactions in an organism can be conceptualised as a network, and is termed the transcriptional regulatory network. 1–11 In such a network, nodes represent genes and edges represent regulatory interactions. Studies on the transcriptional regula- tory network at an abstract level have shown that they have architectures resembling scale-free net- works, with striking structural and topological similarity to other networks from biological and non-biological systems. They are characterized by the recurrence of small patterns of interconnections, called network motifs, 3–5,12–16 which were first defined in Escherichia coli, 3 and were subsequently found in yeast and other organisms. 2,4,12 Even though the general structural properties of transcriptional networks are well understood, there are several fundamental questions regarding the provenance and evolution of transcriptional regu- latory networks that remain unanswered: What are the trends of conservation of transcription factors, 0022-2836/$ - see front matter q 2006 Published by Elsevier Ltd. Abbreviation used: LSI, lifestyle similarity index. E-mail addresses of the corresponding authors: madanm@mrc-lmb.cam.ac.uk; aravind@ncbi.nlm.nih.gov ARTICLE IN PRESS doi:10.1016/j.jmb.2006.02.019 J. Mol. Biol. (2006) xx, 1–20