Complexes in Higher Eukaryotes

The DRIP (Vitamin D3 receptor [VDR] interacting proteins) complex was purified using a VDR ligand-binding domain affinity matrix (Rachez et al., 1998). DRIP is needed for full transcriptional activity of VDR on naked DNA templates in vitro. Another complex, ARC (activator-recruited cofactor), was identified as a complex that enhances transcription activation by SREBP-la, VP16, and the p65 subunit of NF-kappaB using chromatin-assembled DNA templates (Naar et al., 1999). Characterization of the subunits of DRIP and ARC showed that the two complexes are highly related—if not identical—to each other and also to the TRAP/SMCC complexes (Rachez et al., 1999). 

TRAP/SMCC, DRIP, ARC, and human Mediator are virtually identical in their subunit composition (Malik and Roeder, 2000). Another set of Mediator-like complexes has also been isolated that appear to correspond to a submodule of the larger Mediator. This has led to speculations that the Mediator might exist in two separate forms. One of these smaller complexes is PC2, a component of the coactivator fraction USA (Malik et al., 

2000 Meisterernst et al., 1991). PC2 can support activated transcription in vitro, but only in the presence of two other cofactors, PC3/topoisome-rase I and PC4. Two other small Mediator complexes, CRSP (cofactor required for Spl) and mouse Mediator, were both identified using a biochemical fractionation (Jiang et al., 1998 Ryu et aL, 1999). Interestingly, CRSP and PC2 could only support activation in the presence of TAFs (Malik et al., 2000 Ryu et al., 1999), This could indicate a yet-to-be-defined functional relationship between TAFs and the smaller form of Mediator. Another small Mediator complex is NAT (negative regulator of activated transcription). In contrast to the other Mediator complexes, NAT displayed an inhibitory effect on transcription in vitro (Sun et al., 1998). 

The function of individual metazoan Mediator subunits has been studied by different methods that is, by RNA interference (RNAi) and chemical mutagenesis in C. elegans, P insertions in Drosophila, homologous recombination in mouse, and studying spontaneous mutations in human cells (Ito et al., 2000 Kwon et al., 1999 Nilsson et al., 2000 Philibert et al., 1998 Singh and Han, 1995 Spradling et al., 1999 Tudor et al., 1999 Zhu et al., 2000). 

Two Mediator subunits, Med220/TRAP220 and Srb7, have been studied via inactivation of the corresponding genes in mice. In the Srb7 study, heterozygous ES cells and animals showed no phenotype (Tudor et al., 

---

Hashimoto T (2003) Plastid division its origins and evolution. Int Rev Cytol 222 63-98 Iyer LM, Leipe D, Koonin EV, Aravind L (2004) Evolutionary history and higher order classification of AAA+ ATPases. J Struct Biol 146 11-31 Javaux EJ, Knoll AH, Walter MR (2001) Morphological and ecological complexity in early eukaryotic ecosystems. Nature 412 66-69... 

Making the link from structural organization is sometimes considered trivial. It is not, however. The intracellular structures themselves are not constants but depend on the effect of a multimde of cooperating non-equilibrium processes. This is clear for the supercoiling state of prokaryotic DNA (Snoep et al. 2002), but also for the complex structure of chromatin in higher eukaryotes. 

The structural information currently available su ests that p-propellers can act as a scaffold for multiple protein interactions or form a monovalent interaction surface. In cases where the P-propellers are components of ancient protein complexes such as the Atp2/3 complex or het-erotrimeric G-proteins, multiple interactions have evolved. However, other WD- and Kelch-repeat proteins, notably the ubiquidn E3 ligase substrate adaptors, bind only one or several similar sequences. This may explain the expansion of WD- and Kelch-repeat proteins in higher eukaryotes the P-propellers form a r d structural domain, perhaps providing a stable platform on which the surface loops can evolve to form distinct protein-protein interaction domains. 

The 3 -end of an eukaryotic mRNA is formed post-transcriptionally by endonucleolytic cleavage downstream of the coding sequence followed by extension of the upstream fragment by approximately 200 adenylate residues. In higher eukaryotes the specificity of the reaction depends on two conserved sequences in the pre-mRNA a highly conserved AAUAAA 10-30 nucleotides upstream from the cleavage site and a G/U-rich sequence within 50 nucleotides downstream from this site. The reaction requires a complex set of nuclear proteins, and a reconstituted polyadenylation reaction from pure proteins has not yet been achieved. However, processing of... 

In higher eukaryotes, a complex protein structure called the kinetochore assembles at centromeres and associates with multiple mitotic spindle fibers during mitosis. Homologs of most of the centromeric proteins found in the yeasts occur in humans and other higher eukaryotes and are thought to be components of kinetochores. The role of the centromere and proteins that bind to it in the segregation of sister chromatids during mitosis is described in Chapters 20 and 21. 

---