DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation.

MADS domain proteins are members of a highly conserved family found in all eukaryotes. Genetic studies clearly indicate that many plant MADS domain proteins have different regulatory functions in flower development, yet they share a highly conserved DNA binding domain and can bind to very similar sequences. How, then, can these MADS box genes confer their specific functions? Here, we describe results from DNA binding studies of AGL1 and AGL2 (for AGAMOUS-like), two Arabidopsis MADS domain proteins that are preferentially expressed in flowers. We demonstrate that both proteins are sequence-specific DNA binding proteins and show that each binding consensus has distinct features, suggestion a mechanism for specificity. In addition, we show that the proteins with more similar amino acid sequences have more similar binding sequences. We also found that AGL2 binds to DNA in vitro as a dimer and determined the region of AGL2 that is sufficient for DNA binding and dimerization. Finally, we show that several plant MADS domain proteins can bind to DNA either as homodimers or as heterodimers, suggesting that the number of different regulators could be much greater than the number of MADS box genes.     

拟黑多刺蚁肌细胞增强因子2(MEF2)生物信息学分析

应用NCBI上的常用程序、ExPASy在线核苷酸序列分析工具、CBS生物学序列分析工具及SABLE在线分析软件等对拟黑多刺蚁Polyrhachi svicina Roger肌细胞增强因子2(PvMEF2)进行了生物信息学分析,获得了PvMEF2因子的序列特征及理化性质,并对其结构和功能结构域进行了预测。结果表明PvMEF2因子具有与已知种类MEF2因子较高一致性的MADS和MEF2结构域,并且理化性质和二级结构、三级结构等与果蝇该因子类似,反映了PvMEF2可能是参与拟黑多刺蚁肌肉发生调控的重要因子。     

Developmental regulation of the Drosophila tropomyosin I (TmI) gene is controlled by a muscle activator enhancer region that contains multiple cis-elements and binding sites for multiple proteins

Developmental gene regulation in vertebrate somatic muscles involves the cooperative interaction of MEF2 (myocyte-specific enhancer-binding factor 2) and members of the b-HLH (basic helix-loop-helix) family of myogenic factors. Until recently, however, nothing was known about the factors that control the developmental regulation of muscle genes during embryogenesis in Drosophila. The Drosophila Tropomyosin I (TmI) gene contains a proximal and distal muscle enhancer within the first intron that regulates its expression in embryonic/larval and adult muscles. We have recently shown that the 355-bp proximal enhancer contains a binding site for the Drosophila homologue of vertebrate MEF2 and that MEF2 acts cooperatively with a basal level muscle activator region to direct high level muscle expression in transgenic flies. The 92-bp muscle activator region, however, does not contain any consensus E-box (CANNTG) binding site sequences for b-HLH myogenic factors, suggesting the MEF2 may interact with other factors to regulate muscle genes in Drosophila. In this study we have used mutation analysis and germ-line transformation to analyze the cis-acting elements within the muscle activator region that regulate its expression in transgenic flies. We have identified a 71-bp region that is sufficient for low basal level temporal- and muscle-specific expression in the embryo, larva, and adult. Substitution mutations within the muscle activator region have identified several cis-element regions spanning 60-bp that are required for either full or partial muscle activator function. An analysis of proteins that bind to this region by gel mobility shift assay and copper nuclease footprinting has allowed us to identify the sites in this region at which multiple proteins complex and interact. We propose that these cis-elements and the proteins that they bind regulate muscle activator function and together with MEF2 are capable of regulating high level muscle expression. Dev. Genet. 20:297–306, 1997. © 1997 Wiley-Liss, Inc.     

Swapping functional specificity of a MADS box protein: residues required for Arg80 regulation of arginine metabolism

Arg80 and Mcm1, two members of the MADS box family of DNA-binding proteins, regulate the metabolism of arginine in association with Arg81, the arginine sensor. In spite of the high degree of sequence conservation between the MADS box domains of the Arg80 and Mcm1 proteins (56 of 81 amino acids), these domains are not interchangeable. To determine which amino acids define the specificity of Arg80, we swapped the amino acids in each secondary-structure element of the Arg80 MADS box domain with the corresponding amino acids of Mcm1 and assayed the ability of these chimeras to regulate arginine-metabolic genes in place of the wild-type Arg80. Also performed was the converse experiment in which each variant residue in the Mcm1 MADS box domain was swapped with the corresponding residue of Arg80 in the context of an Arg80-Mcm1 fusion protein. We show that multiple regions of Arg80 are important for its function. Interestingly, the residues which have important roles in determining the specificity of Arg80 are not those which could contact the DNA but are residues that are likely to be involved in protein interactions. Many of these residues are clustered on one side of the protein, which could serve as an interface for interaction with Arg81 or Mcm1. This interface is distinct from the region used by the Mcm1 and human serum response factor MADS box proteins to interact with their cofactors. It is possible that this alternative interface is used by other MADS box proteins to interact with their cofactors.