BMC Genomics 2004, 5:69 http://www.biomedcentral.com/1471-2164/5/69 PfCRK1 from Plasmodium. Once again, no kinases from either Trypanosoma or Giardia show any phylogenetic affinity to this group. Discussion A suggestion of co-evolution between the RNAP II CTD and CTD kinases At least five CDKs have the capacity to phosphorylate RNAP II CTD repeats in vitro or in vivo [25-28]. Kinases that appear to be related closely to CDK1, which can phosphorylate the CTD in vitro, are present in all organ- isms sampled; however, it is not clear that CDK1 interacts with the CTD in vivo or is essential for CTD function. CDK2 was found only in human and Drosophila and, based on our analyses from a diverse group of eukaryotes, appears to be derived from within a larger CDK1 family. In any case, according to its restricted phylogenetic distri- bution, possible CTD/CDK2 interactions cannot explain the conservation of CTD structure in diverse members of the "CTD-clade." Evolutionary investigations of RPB1 sequences show that canonical CTD heptads are conserved strongly in only a subset of eukaryotic groups, all apparently descended from a single common ancestor [31]. This "CTD-clade" is composed of animals, plants, fungi, and related protistan groups, including microsporidians, chytridiomycetes, choanoflagellates and slime molds (Fig. 2). A handful of organisms that do not fall inside the "CTD-clade" do have tandemly repeated C-terminal heptads. For example, RPB1 from Plasmodium falciparum contains a short set of seven tandem C-terminal repeats. Based on codon usage and comparative alignment with sequences from other Plasmodium species, these heptads are best explained by a recent tandem duplication of a single heptad motif in P. falciparum or its immediate ancestor [31]. No other api- complexan RPB1 contains tandemly repeated heptads, nor does the nearest evolutionary relative of the apicom- plexans (Fig. 2). Although vestigial or convergent heptad repeats are found in a few organisms scattered across the eukaryotic evolutionary tree, strong stabilizing selection on CTD structure appears to be restricted to those eukary- otic lineages found in the "CTD-clade" (Fig. 2). In our analyses of CDKs, members of this "CTD-clade" are precisely the same eukaryotes to which clear orthologs of CDK7 and CDK8 are restricted. When sequences recov- ered from additional but incomplete eukaryotic genomes are included in phylogenetic analyses, distribution of these two kinases remains tightly correlated with strong conservation of canonical CTD repeats (see additional file 1). Moreover, unlike CDK1, the primary characterized function of both of these kinases is to mediate RNAP IIA/ IIO cycling through reversible phosphorylation of CTD residues [19-24]. Taken together, these findings suggest that the RNAP II CTD has undergone a co-evolutionary process with CDK7 and CDK8. If phylogenetic results based on CDK and RPB1 sequences reflect evolutionary history, the inference of a "CTD-clade" in both sets of analyses suggests that CDK7 and 8 originated as part of a major shift in the mechanics of RNAP II transcription in the ancestor of the "CTD-clade" [31]. It was in that ancestor that reversible phosphorylation of the CTD became a central organizing principle for regulating the transcription cycle, and laid the foundation for more complicated mechanisms of transcriptional control in these organisms. Such a pro- found shift in the mechanics of RNAP II transcription would explain why the CTD is conserved so strongly in members of the CTD-clade, but not in many other eukary- otic lineages [31]. In this scenario, other known or puta- tive CTD kinases (certainly CDK1 and apparently CDK9) originated before canalization of a CTD-based RNAP II transcription cycle, and were adapted later as CTD kinases. It also is possible that the co-evolution inferred from com- parisons of the phylogenetic distribution of RPB1 and CDKs7/8 does not reflect the pattern of evolutionary his- tory but, instead, results from functional constraints driven by CTD/CDK interactions. Both GlCAKlike from Giardia and PfMrk from Plasmodium have been suggested previously to be orthologs of CDK7 [36,38]; these hypotheses are rejected strongly by our phylogenetic anal- yses. Assuming these kinases really are CDK7s, then their failure to cluster with other orthologs must be due to phy- logenetic artifacts, frequently referred to as "long-branch attraction" [39], that can be common when rates of evolu- tion vary dramatically among sequences. The large amounts of sequence divergence of PfMrk and GlCAKlike from other CDK7s, along with a complete degeneration of the CTD in Giardia species and apicomplexans as a group, are unlikely to coincidental. It is possible that those organisms retaining a RNAP II transcription cycle medi- ated by CDK7 and 8 kinase activity form distinct clades, in both RPB1 and kinase derived trees, because both sets of proteins share parallel modes of evolution driven by their physical interactions. In this case, the observation of co- evolution between the CTD and CTD-directed kinases need not have a phylogenetic basis, only a functional one. Most putative CDKs from Giardia and Trypanosoma, and several from Plasmodium, do not associate strongly with any established CDK family. It is reasonable to assume that at least some of these kinases are orthologs of defined CDK groups, but have diverged to the point that they are not recognizable using sequence-based phylogenetic methods. Although such a scenario may have disturbing implications for the use of these methods across broad evolutionary distances, particularly when functional Page 8 of 13 (page number not for citation purposes)