The 2.5 A resolution structure of a cocrystal containing the paired domain from the Drosophila paired (prd) protein and a 15 bp site shows structurally independent N-terminal and C-terminal subdomains. Each of these domains contains a helical region resembling the homeodomain and the Hin recombinase. The N-terminal domain makes extensive DNA contacts, using a novel beta turn motif that binds in the minor groove and a helix-turn-helix unit with a docking arrangement surprisingly similar to that of the lambda repressor. The C-terminal domain is not essential for prd binding and does not contact the optimized site. All known developmental missense mutations in the paired box of mammalian Pax genes map to the N-terminal subdomain, and most of them are found at the protein-DNA interface.

We report the identification of a Drosophila Pax gene, eye gone (eyg), which is required for eye development. Loss-offunction eyg mutations cause reduction or absence of the eye. Similar to the Pax6 eyeless (ey) gene, ectopic expression of eyg induces extra eye formation, but at sites different from those induced by ey. Several lines of evidence suggest that eyg and ey act cooperatively: (1) eyg expression is not regulated by ey, nor does it regulate ey expression, (2) eyginduced ectopic morphogenetic furrow formation does not require ey, nor does ey-induced ectopic eye production require eyg, (3) eyg and ey can partially substitute for the function of the other, and (4) coexpression of eyg and ey has a synergistic enhancement of ectopic eye formation. Our results also show that eyg has two major functions: to promote cell proliferation in the eye disc and to promote eye development through suppression of wg transcription.

Nearly all metazoan homeodomains (HDs) possess DNA binding targets that are related by the presence of a TAAT sequence. We use an in vitro genetic DNA binding site selection assay to refine our understanding of the amino acid determinants for the recognition of the TAAT site. Superimposed upon the conserved ability of metazoan HDs to recognize a TAAT core is a difference in their preference for the bases that lie immediately 3' to it. Amino acid position 50 of the HD has been shown to discriminate among these base pairs, and structural studies have suggested that watermediated hydrogen bonds and van der Waals contacts underlie for this ability. Here, we show that each of six amino acids tested at position 50 can confer a distinct DNA binding specificity.The homeodomain (HD) is one of the most common DNA binding motifs in eukaryotic transcriptional regulators (1). Structural studies have shown that the HD is composed of three a-helices and a flexible N-terminal arm (2). The third helix of the HD inserts itself into the major groove of the DNA and is, therefore, called the recognition helix (3, 4). In addition, the N-terminal arm of the HD inserts into the minor groove of the DNA, making several contacts with the bases (4, 5).Despite the vast number of different biological functions of homeoproteins, nearly all homeodomains from metazoans bind to sequences containing a TAAT core motif (6-8). The anatomical descriptions of HD-DNA interactions derived from structural studies suggest a mechanism by which the conserved TAAT motif is recognized by HDs (3,4,[9][10][11]. In this report, we analyze the optimal DNA binding specificities of several HDs. By combining these results with previously reported DNA site selection experiments, we derive an amino acid consensus sequence for the recognition of the TAAT motif. This consensus is similar to but distinct from those derived previously using different assumptions or more limited data sets. Our consensus includes five amino acid positions that interact with the base pairs of the TAAT motif. Two of these amino acid positions, which map to the N-terminal arm, can form alternative interaction with the same base pair.Although most metazoan HDs share a preference for the TAAT motif, they can differ from each other in their preference for the bases immediately 3' to this core (8,(12)(13)(14)(15) (20). The site, like that of Ftz, is composed of the nearly universal TAAT motif, but the bases 3' to the TAAT differ. This difference has been shown to be due to the presence of a lysine, rather than a glutamine, at position 50. The lysine specifies the CC sequence immediately 3' to the TAAT motif (8,(12)(13)(14)(15).

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.Pax genes play key roles in regulating developmental processes and are characterized by a conserved motif, the paired box, which encodes the paired domain (PD; ref. 1). The PD is a DNA-binding domain (2) that is composed of two separable and independent subdomains, the N-terminal PAI and the C-terminal RED domains (3, 4), each containing a helix-turnhelix (HTH) motif (5). The PAI domain seems to be the most critical part of the PD (2, 5), because it is necessary and sufficient for DNA binding in vitro (2-4, 6). In fact, the Drosophila Prd protein does not need the RED domain to confer full function to the protein in vivo (7,8). There are, however, situations in which the RED domain may play a role (3). The bipartite organization of the PD allows for the recognition of composite sites by both PAI and RED domain. Furthermore, there is an isoform of the Pax-6 protein (Pax-6 5a) that contains, in the middle of the recognition helix of the PAI domain, an 11-residue insertion that inactivates DNA binding to sequences normally bound by the Pax-6 PD (6). Pax-6 5a, however, is able to bind to a recently described sequence called 5aCON (6). Other Pax proteins also contain insertions in the PAI domain that inactivate DNA binding and uncover RED-domain DNA-binding activity (9). However, to date, there has been no report of a Pax protein containing only a PAI or a RED domain.About half of the known Pax genes (1, 10-16) also encode a second DNA-binding domain, a Prd-class homeodomain (HD). The Prd-class HDs recognize DNA through cooperative dimerization mediated by the HD (17, 18). There are several Prd HD subclasses that are...

The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs, the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

The Drosophila pair-rule gene paired is required for the correct expression of the segment polarity genes wingless, engrailed and gooseberry. It encodes a protein containing three conserved motifs: a homeodomain (HD), a paired domain (PD) and a PRD (His/Pro) repeat. We use a rescue assay in which paired (or a mutated version of paired in which the functions of the conserved motifs have been altered) is expressed under the control of its own promoter, in the absence of endogenous paired, to dissect the Paired protein in vivo. We show that both the HD and the N- terminal subdomain of the PD (PAI domain) are absolutely required within the same molecule for normal paired function. In contrast, the conserved C-terminal subdomain of the PD (RED domain) appears to be dispensable. Furthermore, although a mutation abolishing the ability of the homeodomain to dimerize results in an impaired Paired molecule, this molecule is nonetheless able to mediate a high degree of rescue. Finally, a paired transgene lacking the PRD repeat is functionally impaired, but still able to rescue to viability. We conclude that, while Prd can use its DNA-binding domains combinatorially in order to achieve different DNA-binding specificities, its principal binding mode requires a cooperative interaction between the PAI domain and the homeodomain.