br Acknowledgements This work was supported in

Acknowledgements This work was supported, in part, by NIH grant R01-GM071760.
Introduction The MADS box genes encode a eukaryotic family of transcriptional regulators involved in diverse and important biological functions. This class of proteins has been identified in yeasts, plants, insects, nematodes, lower vertebrates and mammals. To date, the plant databases reveal the existence of several hundreds of putative MADS box genes. These proteins contain a conserved DNA binding and dimerization domain named the MADS box (Schwarz-Sommer et al., 1990) after the five founding members of the family: Mcm1(yeast) (Passmore et al., 1989), Arg80 (yeast) (Dubois et al., 1987) or Agamous (plant) (Yanofsky et al., 1990), Deficiens (plant) (Sommer et al., 1990) and SRF (human) (Norman et al., 1988). In animal and fungi, two distinct types of MADS box genes have been identified, the SRF-like (type I) and MEF2-like (type II) classes (Shore and Sharrocks, 1995). These MEF2-like amino 88 3 sequences are more closely related to most plant MADS-domain sequences, suggesting that at least one gene-duplication event occurred before the divergence of plant and animals (Alvarez-Buylla et al., 2000). However, a group of Arabidopsis MADS domain sequences seem to share a more closely related ancestor with the SRF-like sequences from animals and fungi than with other plant MADS-domain sequences. Thus, both type I and type II MADS box proteins can be found in each kingdom. The organization of type I and type II MADS box proteins is presented in Fig. 1. Type I and type II proteins can be classified in subfamilies on the basis of shared sequence similarity between their extensions C-terminal to the highly conserved MADS domain. These domains are referred to as SAM in fungi and animal SRF-like proteins, as MEF2 in fungi and animal MEF2-like proteins and as I, K and C in plant type II proteins Mueller and Nordheim, 1991, Sharrocks et al., 1993, Riechmann et al., 1996, Riechmann and Meyerowitz, 1997. These C-terminal extensions may add in specifying dimerization between members of the same subfamily, which could explain why attempts to form dimers with MADS box proteins possessing alternative C-terminal extensions have been unsuccessful (Pollock and Treisman, 1991). MADS box family members generally recognize AT-rich consensus sequences, with a highly conserved core of 10 bp: CC(A/T)6GG known as CArG box is the binding site of SRF-like proteins (Treisman, 1990), and CTA(A/T)4TAG is the binding site of MEF2-like proteins (Pollock and Treisman, 1991)(Fig. 2c and e). These DNA consensus sequences were used to generate crystal structures of SRF, SRF-SAP1, Mcm1–α2 and MEF2A in complex with DNA Pellegrini et al., 1995, Tan and Richmond, 1998, Huang et al., 2000, Santelli and Richmond, 2000, Hassler and Richmond, 2001, Mo et al., 2001. Comparison of these structures revealed that the conformation of the MADS box domains and many of the DNA contacts were remarkably conserved between the yeast and human proteins. As shown in Fig. 2, the MADS box domain of SRF or MEF2A forms a protein dimer composed of three layers. The first layer consists of antiparallel coiled-coil α helices, one from each monomer, positioned above the center of the recognition site. The middle layer consists of a hydrophobic four-stranded β sheet, formed from two β strands from each monomer and is aligned parallel to the coiled-coil α helices. The top layer consists of an extended coil region followed by α helix made of three turns. Binding of MADS box proteins to DNA induces DNA bending, however, with different amplitudes. SRF, Mcm1 and some plant MADS box proteins introduce significant DNA distortion. In contrast, MEF2A and the yeast proteins Rlm1 and Smp1 induce minimal DNA bending West et al., 1997, West et al., 1998, West and Sharrocks, 1999. DNA bending induced by SRF and MEF2A is illustrated in Fig. 2b and d. DNA bending is thought to play a role in the DNA recognition process and in determining the correct architecture of nucleoprotein complexes at promoters and enhancers.