1) Natural history and classification of the Helix-turn-helix domains. The helix-turn-helix (HTH) domain is a common denominator in basal and specific transcription factors from the three super-kingdoms of life. At its core, the domain comprises of an open tri-helical bundle, which typically binds DNA with the 3rd helix. Drawing on the wealth of data that has accumulated over two decades since the discovery of the domain, we present an overview of the natural history of the HTH domain from the viewpoint of structural analysis and comparative genomics. In structural terms, the HTH domains have developed several elaborations on the basic 3-helical core, such as the tetra-helical bundle, the winged-helix and the ribbon-helix-helix type configurations. In functional terms, the HTH domains are present in the most prevalent transcription factors of all prokaryotic genomes and some eukaryotic genomes. They have been recruited to a wide range of functions beyond transcription regulation, which include DNA repair and replication, RNA metabolism and protein-protein interactions in diverse signaling contexts. Beyond their basic role in mediating macromolecular interactions, the HTH domains have also been incorporated into the catalytic domains of diverse enzymes. We discuss the general domain architectural themes that have arisen amongst the HTH domains as a result of their recruitment to these diverse functions. We present a natural classification, higher-order relationships and phyletic pattern analysis of all the major families of HTH domains. This reconstruction suggests that there were at least 6-11 different HTH domains in the last universal common ancestor of all life forms, which covered much of the structural diversity and part of the functional versatility of the extant representatives of this domain. In prokaryotes the total number of HTH domains per genome shows a strong power-equation type scaling with the gene number per genome. However, the HTH domains in two-component signaling pathways show a linear scaling with gene number, in contrast to the non-linear scaling of HTH domains in single-component systems and sigma factors. These observations point to distinct evolutionary forces in the emergence of different signaling systems with HTH transcription factors. The archaea and bacteria share a number of ancient families of specific HTH transcription factors. However, they do not share any orthologous HTH proteins in the basal transcription apparatus. This differential relationship of their basal and specific transcriptional machinery poses an apparent conundrum regarding the origins of their transcription apparatus. 2) Origin of acetylcholine-receptor type ligand gated channels of animals. Acetylcholine receptor type ligand-gated ion channels (ART-LGIC; also known as Cys-loop receptors) are a superfamily of proteins that include the receptors for major neurotransmitters such as acetylcholine, serotonin, glycine, GABA, glutamate and histamine, and for Zn2+ ions. They play a central role in fast synaptic signaling in animal nervous systems and so far have not been found outside of the Metazoa. We have identified homologs of ART-LGICs in several bacteria and a single archaeal genus, Methanosarcina. The homology between the animal receptors and the prokaryotic homologs spans the entire length of the former, including both the ligand-binding and channel-forming transmembrane domains. A sequence-structure analysis using the structure of Lymnaea stagnalis acetylcholine-binding protein and the newly detected prokaryotic versions indicates the presence of at least one aromatic residue in the ligand-binding boxes of almost all representatives of the superfamily. Investigation of the domain architectures of the bacterial forms shows that they may often show fusions with other small-molecule-binding domains, such as the periplasmic binding protein superfamily I (PBP-I), Cache and MCP-N domains. Some of the bacterial forms also occur in predicted operons with the genes of the PBP-II superfamily and the Cache domains. Analysis of phyletic patterns suggests that the ART-LGICs are currently absent in all other eukaryotic lineages except animals. Moreover, phylogenetic analysis and conserved sequence motifs also suggest that a subset of the bacterial forms is closer to the metazoan forms. From the information from the bacterial forms we infer that cation-pi or hydrophobic interactions with the ligand are likely to be a pervasive feature of the entire superfamily, even though the individual residues involved in the process may vary. The conservation pattern in the channel-forming transmembrane domains also suggests similar channel-gating mechanisms in the prokaryotic versions. From the distribution of charged residues in the prokaryotic M2 transmembrane segments, we expect that there will be examples of both cation and anion selectivity within the prokaryotic members. Contextual connections suggest that the prokaryotic forms may function as chemotactic receptors for low molecular weight solutes. The phyletic patterns and phylogenetic relationships suggest the possibility that the metazoan receptors emerged through an early lateral transfer from a prokaryotic source, before the divergence of extant metazoan lineages. 3) Apicomplexan transcription factors. The comparative genomics of apicomplexans, such as the malarial parasite Plasmodium, the cattle parasite Theileria and the emerging human parasite Cryptosporidium, have suggested an unexpected paucity of specific transcription factors (TFs) with DNA binding domains that are closely related to those found in the major families of TFs from other eukaryotes. This apparent lack of specific TFs is paradoxical, given that the apicomplexans show a complex developmental cycle in one or more hosts and a reproducible pattern of differential gene expression in course of this cycle. Using sensitive sequence profile searches, we show that the apicomplexans possess a lineage-specific expansion of a novel family of proteins with a version of the AP2 (Apetala2)-integrase DNA binding domain, which is present in numerous plant TFs. About 20-27 members of this apicomplexan AP2 (ApiAP2) family are encoded in different apicomplexan genomes, wth each protein containing one to four copies of the AP2 DNA binding domain. Using gene expression data from Plasmodium falciparum, we show that guilds of ApiAP2 genes are expressed in different stages of intraerythrocytic development. By analogy to the plant AP2 proteins and based on the expression patterns, we predict that the ApiAP2 proteins are likely to function as previously unknown specific TFs in the apicomplexans and regulate the progression of their developmental cycle. In addition to the ApiAP2 family, we also identified two other novel families of AP2 DNA binding domains in bacteria and transposons. Using structure similarity searches, we also identified divergent versions of the AP2-integrase DNA binding domain fold in the DNA binding region of the PI-SceI homing endonuclease and the C-terminal domain of the pleckstrin homology (PH) domain-like modules of eukaryotes. Integrating these findings, we present a reconstruction of the evolutionary scenario of the AP2-integrase DNA binding domain fold, which suggests that it underwent multiple independent combinations with different types of mobile endonucleases or recombinases. It appears that the eukaryotic versions have emerged from versions of the domain associated with mobile elements, followed by independent lineage-specific expansions, which accompanied their recruitment to transcription regulation functions.
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