Publications

1995
Kim, J, Irvine KD, Carroll SB.  1995.  Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing Drosophila wing. Cell. 82:795-802. AbstractWebsite
Appendage formation in insects and vertebrates depends upon signals from both the anterior-posterior and dorsal-ventral (DV) axes. In Drosophila, wing formation is organized symmetrically around the DV boundary of the growing wing imaginal disc and requires interactions between dorsal and ventral cells. Compartmentalization of the wing disc, dorsal cell behavior, and the expression of two dorsally expressed putative signaling molecules, fringe (fng) and Serrate (Ser), are regulated by the apterous selector gene. Here, we demonstrate that fng and Ser have distinct roles in a novel cell recognition and signal induction process. fng serves as a boundary-determining molecule such that Ser is induced wherever cells expressing fng and cells not expressing fng are juxtaposed. Ser in turn triggers the expression of genes involved in wing growth and patterning on both sides of the DV boundary.
1996
Tang, H, Kim Y, Severinov K, Goldfarb A, Ebright RH.  1996.  Escherichia coli RNA polymerase holoenzyme: rapid reconstitution from recombinant alpha, beta, beta', and sigma subunits.. Methods in enzymology. 273:130-4.
Heyduk, T, Ma Y, Tang H, Ebright RH.  1996.  Fluorescence anisotropy: rapid, quantitative assay for protein-DNA and protein-protein interaction.. Methods in enzymology. 274:492-503.
Dumoulin, P, Ebright RH, Knegtel R, Kaptein R, Granger-Schnarr M, Schnarr M.  1996.  Structure of the LexA repressor-DNA complex probed by affinity cleavage and affinity photo-cross-linking.. Biochemistry. 35(14):4279-86. Abstract
The structure of the complex of full-length Escherichia coli LexA repressor with a consensus operator DNA fragment has been probed by affinity photo-cross-linking and affinity cleavage. These methods allow the determination of approximate intermolecular distances between a given protein residue and a base or sugar moiety within the operator. In a first step unique cysteine residues were introduced in positions 7, 28, 38, or 52 of the protein. In all four cases, the original amino acid was an arginine. The four amino acids in these positions were expected to be situated on the surface of LexA interacting with DNA, as inferred from the structure of the LexA DNA binding domain [Fogh et al. (1994) EMBO J. 13, 3936-3944]. In a second step, these unique cysteine side chains of the purified proteins were chemically modified either with 4-azidophenacyl bromide or with S-(2-pyridylthio)cysteaminyl-EDTA. The first set of derivatives gives rise to UV-induced cross-linking which may be revealed by alkali/heat treatment; the second leads to direct DNA cleavage in the proximity of the derivatized amino acid. To reduce hydroxyl radical diffusion, the EDTA-iron cleavage reactions were done in the presence of high amounts of glycerol. The results indicate that amino acids 7 and 52 are near nucleotide pairs 8-12 of the operator and that amino acids 28 and 36 of LexA are near nucleotide pairs 5-8 of the operator. The results unambiguously define the orientation of the LexA DNA binding domain relative to the operator and provide support for the model of the LexA-operator complex proposed by Knegtel et al. [(1995) Proteins 21, 226-236]. Ethylation interference experiments further suggest that Arg-7 contacts the phosphate group between nucleotides 8 and 9 as predicted by the model.
Niu, W, Kim Y, Tau G, Heyduk T, Ebright RH.  1996.  Transcription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase.. Cell. 87(6):1123-34. Abstract
At Class II catabolite activator protein (CAP)-dependent promoters, CAP activates transcription from a DNA site overlapping the DNA site for RNA polymerase. We show that transcription activation at Class II CAP-dependent promoters requires not only the previously characterized interaction between an activating region of CAP and the RNA polymerase alpha subunit C-terminal domain, but also an interaction between a second, promoter-class-specific activating region of CAP and the RNA polymerase alpha subunit N-terminal domain. We further show that the two interactions affect different steps in transcription initiation. Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how an activator can make multiple interactions with the transcription machinery, each interaction being responsible for a specific mechanistic consequence.
Tang, H, Sun X, Reinberg D, Ebright RH.  1996.  Protein-protein interactions in eukaryotic transcription initiation: structure of the preinitiation complex.. Proceedings of the National Academy of Sciences of the United States of America. 93(3):1119-24. Abstract
We have used alanine scanning to analyze protein-protein interactions by human TATA-element binding protein (TBP) within the transcription preinitiation complex. The results indicate that TBP interacts with RNA polymerase II and general transcription factors IIA, IIB, and IIF within the functional transcription preinitiation complex and define the determinants of TBP for each of these interactions. The results permit construction of a model for the structure of the preinitiation complex.
Gaal, T, Ross W, Blatter EE, Tang H, Jia X, Krishnan VV, Assa-Munt N, Ebright RH, Gourse RL.  1996.  DNA-binding determinants of the alpha subunit of RNA polymerase: novel DNA-binding domain architecture.. Genes & development. 10(1):16-26. Abstract
The Escherichia coli RNA polymerase alpha-subunit binds through its carboxy-terminal domain (alpha CTD) to a recognition element, the upstream (UP) element, in certain promoters. We used genetic and biochemical techniques to identify the residues in alpha CTD important for UP-element-dependent transcription and DNA binding. These residues occur in two regions of alpha CTD, close to but distinct from, residues important for interactions with certain transcription activators. We used NMR spectroscopy to determine the secondary structure of alpha CTD, alpha CTD contains a nonstandard helix followed by four alpha-helices. The two regions of alpha CTD important for DNA binding correspond to the first alpha-helix and the loop between the third and fourth alpha-helices. The alpha CTD DNA-binding domain architecture is unlike any DNA-binding architecture identified to date, and we propose that alpha CTD has a novel mode of interaction with DNA. Our results suggest models for alpha CTD-DNA and alpha CTD-DNA-activator interactions during transcription initiation.
Parkinson, G, Wilson C, Gunasekera A, Ebright YW, Ebright RE, Berman HM.  1996.  Structure of the CAP-DNA complex at 2.5 angstroms resolution: a complete picture of the protein-DNA interface.. Journal of molecular biology. 260(3):395-408. Abstract
The crystallographic structure of the CAP-DNA complex at 3.0 A resolution has been reported previously. For technical reasons, the reported structure had been determined using a gapped DNA molecule lacking two phosphates important for CAP-DNA interaction. In this work, we report the crystallographic structure of the CAP-DNA complex at 2.5 A resolution using a DNA molecule having all phosphates important for CAP-DNA interaction. The present resolution permits unambiguous identification of amino acid-base and amino acid-phosphate hydrogen bonded contacts in the CAP-DNA complex. In addition, the present resolution permits accurate definition of the kinked DNA conformation in the CAP-DNA complex.
Sheehan, B, Klarsfeld A, Ebright R, Cossart P.  1996.  A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence.. Molecular microbiology. 20(4):785-97. Abstract
PrfA, the regulator of virulence-gene expression in the pathogenic bacterium Listeria monocytogenes, displays sequence similarity to members of the CAP-FNR family of transcriptional regulators. To test the functional significance of this similarity, we constructed and analysed substitutions of two amino acids of PrfA predicted to contact DNA, i.e. Ser-184 and Ser-183. Substitution of Ser-184 by Ala reduced DNA binding and virulence-gene activation, and attenuated the virulence in a mouse model of infection. In contrast, substitution of Ser-183 by Ala had the opposite effect in these functional assays. A 17bp DNA sequence, which includes a putative PrfA site, was shown to be sufficient for target-site recognition by PrfA and PrfA-S183A. Our results strongly support the hypothesis that PrfA is a structural and functional homologue of CAP. In addition, they establish a clear correlation between DNA binding by PrfA, virulence-gene activation, and virulence.
Ebright, YW, Chen Y, Kim Y, Ebright RH.  1996.  S-[2-(4-azidosalicylamido)ethylthio]-2-thiopyridine: radioiodinatable, cleavable, photoactivatible cross-linking agent.. Bioconjugate chemistry. 7(3):380-4. Abstract
S-[2-(4-Azidosalicylamido)ethylthio]-2-thiopyridine (AET) contains a 2-thiopyridyl moiety, which permits cysteine-specific incorporation into protein through a cleavable disulfide bond, and a 4-azidosalicylamido moiety, which permits radioiodination and photoactivatible cross-linking. In contrast to the related compound S-[2-[N-[4-(4-azidosalicylamido)butyl]carbomoyl]ethylthio]-2 -thiopyridine [APDP; Zecherle, G., Oleinikov, A., and Traut, R. (1992) Biochemistry 31, 9526], AET contains a relatively short linker arm between the 2-thiopyridyl moiety and the 4-azidosalicylamido moiety. In a previous paper, it was shown that AET could be used in site-specific protein-protein photocross-linking to identify nearest-neighbor protein domains within a multiprotein complex [Chen, Y., Ebright, Y., and Ebright, R. (1994) Science 265, 90]. In this paper, the synthesis, radioiodination, and incorporation into protein of AET are described.
Parkinson, G, Gunasekera A, Vojtechovsky J, Zhang X, Kunkel TA, Berman H, Ebright RH.  1996.  Aromatic hydrogen bond in sequence-specific protein DNA recognition.. Nature structural biology. 3(10):837-41.
Lagrange, T, Kim TK, Orphanides G, Ebright YW, Ebright RH, Reinberg D.  1996.  High-resolution mapping of nucleoprotein complexes by site-specific protein-DNA photocrosslinking: organization of the human TBP-TFIIA-TFIIB-DNA quaternary complex.. Proceedings of the National Academy of Sciences of the United States of America. 93(20):10620-5. Abstract
We have used a novel site-specific protein-DNA photocrosslinking procedure to define the positions of polypeptide chains relative to promoter DNA in binary, ternary, and quaternary complexes containing human TATA-binding protein, human or yeast transcription factor IIA (TFIIA), human transcription factor IIB (TFIIB), and promoter DNA. The results indicate that TFIIA and TFIIB make more extensive interactions with promoter DNA than previously anticipated. TATA-binding protein, TFIIA, and TFIIB surround promoter DNA for two turns of DNA helix and thus may form a "cylindrical clamp" effectively topologically linked to promoter DNA. Our results have implications for the energetics, DNA-sequence-specificity, and pathway of assembly of eukaryotic transcription complexes.
Heyduk, T, Heyduk E, Severinov K, Tang H, Ebright RH.  1996.  Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.. Proceedings of the National Academy of Sciences of the United States of America. 93(19):10162-6. Abstract
Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta (i.e., in alpha 2 beta, alpha 2 beta beta', and alpha 2 beta beta' sigma), and amino acids 175-210 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta' (i.e., in alpha 2 beta beta' and alpha 2 beta beta' sigma). The protected regions are conserved in the alpha homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of alpha for direct functional interaction with beta and beta'. The observed maximal magnitude of protection upon interaction with beta and the observed maximal magnitude of protection upon interaction with beta' both correspond to the expected value for complete protection of one of the two alpha protomers of RNAP (i.e., 50% protection). We propose that only one of the two alpha protomers of RNAP interacts with beta and that only one of the two alpha protomers of RNAP interacts with beta'.
Yingling, JM, Das P, Savage C, Zhang M, Padgett RW, Wang XF.  1996.  Mammalian dwarfins are phosphorylated in response to transforming growth factor-β and are implicated in control of cell growth. Proceedings of the National Academy of Sciences of the United States of America. 93:8940-4. AbstractWebsite
The dwarfin protein family has been genetically implicated in transforming growth factor beta (TGF-beta)-like signaling pathways in Drosophila and Caenorhabditis elegans. To investigate the role of these proteins in mammalian signaling pathways, we have isolated and studied two murine dwarfins, dwarfin-A and dwarfin-C. Using antibodies against dwarfin-A and dwarfin-C, we show that these two dwarfins and an immunogenically related protein, presumably also a dwarfin, are phosphorylated in a time- and dose-dependent manner in response to TGF-beta. Bone morphogenetic protein 2, a TGF-beta superfamily ligand, induces phosphorylation of only the related dwarfin protein. Thus, TGF-beta superfamily members may use overlapping yet distinct dwarfins to mediate their intracellular signals. Furthermore, transient overexpression of either dwarfin-A or dwarfin-C causes growth arrest, implicating the dwarfins in growth regulation. This work provides strong biochemical and preliminary functional evidence that dwarfin-A and dwarfin-C represent prototypic members of a family of mammalian proteins that may serve as mediators of signaling pathways for TGF-beta superfamily members.
Yu, K, Sturtevant MA, Biehs B, François V, Padgett RW, Blackman RK, Bier E.  1996.  The Drosophila decapentaplegic and short gastrulation genes function antagonistically during adult wing vein development. Development (Cambridge, England). 122:4033-44. AbstractWebsite
TGF-beta-related signaling pathways play diverse roles during vertebrate and invertebrate development. A common mechanism for regulating the activity of TGF-beta family members is inhibition by extracellular antagonists. Recently, the Drosophila short gastrulation (sog) gene was shown to encode a predicted diffusible factor which antagonizes signaling mediated by the TGF-beta-like Decapentaplegic (Dpp) pathway in the early blastoderm embryo. sog and dpp, which are among the earliest zygotic genes to be activated, are expressed in complementary dorsal-ventral domains. The opposing actions of sog and dpp in the early embryo have been highly conserved during evolution as their vertebrate counterparts, chordin and BMP-4, function homologously to define neural versus non-neural ectoderm in Xenopus. Here we exploit the genetically sensitive adult wing vein pattern to investigate the generality of the antagonistic relationship between sog and dpp. We show that dpp is expressed in vein primordia during pupal wing development and functions to promote vein formation. In contrast, sog is expressed in complementary intervein cells and suppresses vein formation. sog and dpp function during the same phenocritical periods (i.e. 16-28 hours after pupariation) to influence the vein versus intervein cell fate choice. The conflicting activities of dpp and sog are also revealed by antagonistic dosage-sensitive interactions between these two genes during vein development. Analysis of vein and intervein marker expression in dpp and sog mutant wings suggests that dpp promotes vein fates indirectly by activating the vein gene rhomboid (rho), and that sog functions by blocking an autoactivating Dpp feedback loop. These data support the view that Sog is a dedicated Dpp antagonist.
Savage, C, Das P, Finelli A, Townsend S, Sun C, Baird S, Padgett R.  1996.  Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor β pathway components. Proc Natl Acad Sci U S A. 93:790-794. AbstractWebsite
Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers.
Rongo, C, Lehmann R.  1996.  Regulated synthesis, transport and assembly of the Drosophila germ plasm. Trends Genet. 12:102-9. AbstractWebsite
Germ cells are set aside during early development and, in many organisms (including Drosophila melanogaster, Caenorhabditis elegans and Xenopus laevis), they form in a unique cytoplasm, termed the germ plasm. The germ plasm is synthesized during oogenesis, and the initial polarization of the oocyte is likely to determine where the germ plasm will form within the egg cell. Although we do not know how the fate of germ cells is specified in any organism, recent genetic analysis in Drosophila has identified the TGF-alpha homolog gurken as the signal involved in the initial polarization of the oocyte. These results imply that the limiting steps in the assembly of the germ plasm are localization of the OSK RNA and regulated synthesis of the OSK protein, encoded by oskar, which are components of the germ plasm.
Twombly, V, Blackman RK, Jin H, Graff JM, Padgett RW, Gelbart WM.  1996.  The TGF-beta signaling pathway is essential for Drosophila oogenesis. Development (Cambridge, England). 122:1555-65. AbstractWebsite
We examine roles of signaling by secreted ligands of the TGF-beta family during Drosophila oogenesis. One family member, the DPP ligand encoded by the decapentaplegic (dpp) gene, is required for patterning of anterior eggshell structures. This requirement presumably reflects the expression pattern of dpp in an anterior subset of somatic follicle cells: the centripetally migrating and the nurse cell-associated follicle cells. Similar requirements are also revealed by mutations in the saxophone (sax)-encoded receptor, consistent with the idea that DPP signaling is, at least in part, mediated by the SAX receptor. A loss of germline sax function results in a block in oogenesis associated with egg chamber degeneration and a failure of the transfer of nurse cell contents to the oocyte, indicating that TGF-beta signaling is required for these events. Some phenotypes of sax mutations during oogenesis suggest that SAX responds to at least one other TGF-beta ligand as well in the posterior follicle cells.
Derynck, R, Gelbart WM, Harland RM, Heldin CH, Kern SE, Massagué J, Melton DA, Mlodzik M, Padgett RW, Roberts AB et al..  1996.  Nomenclature: vertebrate mediators of TGFβ family signals. Cell. 87:173.Website
1997
Szeto, L, Fafalios MK, Zhong H, Vershon AK, Broach JR.  1997.  Alpha2p Controls Donor Preference During Mating type Interconversion in Yeast by Inactivating a Recombinational Enhancer of Chromosome IIi. Genes Dev. 11:1899-1911. Abstract
Homothallic strains of Saccharomyces cerevisiae can change mating type as often as every generation by replacing the allele at the MAT locus with a copy of mating type information present at one of two storage loci, HML and HMR, located on either end of chromosome III. Selection of the appropriate donor locus is dictated by a mating type-specific repressor protein, alpha2p: Cells containing alpha2p select HMR, whereas those lacking alpha2p select HML. As a repressor protein, alpha2p binds to DNA cooperatively with the transcriptional activator Mcm1p. Here we show that two alpha2p/Mcm1p-binding sites, DPS1 and DPS2, control donor selection. DPS1 and DPS2 are located approximately 30 kb from the left arm of chromosome III, well removed from HML, HMR, and MAT. Precise deletion of only DPS1 and DPS2 results in random selection of donor loci and in a cells without affecting selection in alpha cells. Reciprocally, deletion of only the alpha2p binding segments in each of these two sites results in selection of the wrong donor loci in alpha cells without affecting preference in a cells. These results suggest that Mcm1p, bound to these two sites in the absence of alpha2p, activates HML as donor. Binding of alpha2p blocks the ability of Mcm1p bound to DPS1 and DPS2 to activate HML, resulting in default selection of HMR as donor. DPS1 and DPS2 also regulate expression of several noncoding RNAs, although deletion of at least one of these RNA loci does not affect donor preference. This suggests that transcriptional activation, rather than transcription of a specific product, is the initiating event in activating the left arm of chromosome III for donor selection.
Gailus-Durner, V, Chintamaneni C, Wilson R, Brill SJ, Vershon AK.  1997.  Analysis of a Meiosis-specific uRS1 Site: Sequence Requirements and Involvement of Replication Protein a. Mol Cell Biol. 17:3536-3546. Abstract
URS1 is a transcriptional repressor site found in the promoters of a wide variety of yeast genes that are induced under stress conditions. In the context of meiotic promoters, URS1 sites act as repressor sequences during mitosis and function as activator sites during meiosis. We have investigated the sequence requirements of the URS1 site of the meiosis-specific HOP1 gene (URS1H) and have found differences compared with a URS1 site from a nonmeiotic gene. We have also observed that the sequence specificity for meiotic activation at this site differs from that for mitotic repression. Base pairs flanking the conserved core sequence enhance meiotic induction but are not required for mitotic repression of HOP1. Electrophoretic mobility shift assays of mitotic and meiotic cell extracts show a complex pattern of DNA-protein complexes, suggesting that several different protein factors bind specifically to the site. We have determined that one of the complexes of URS1H is formed by replication protein A (RPA). Although RPA binds to the double-stranded URS1H site in vitro, it has much higher affinity for single-stranded than for double-stranded URS1H, and one-hybrid assays suggest that RPA does not bind to this site at detectable levels in vivo. In addition, conditional-lethal mutations in RPA were found to have no effect on URS1H-mediated repression. These results suggest that although RPA binds to URS1H in vitro, it does not appear to have a functional role in transcriptional repression through this site in vivo.
Acton, TB, Zhong H, Vershon AK.  1997.  DNA-binding Specificity of Mcm1: Operator Mutations that Alter DNA-bending and Transcriptional Activities by a MADs box Protein. Mol Cell Biol. 17:1881-1889. Abstract
The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins found in such diverse organisms as yeast, plants, flies, and humans. To explore the protein-DNA interactions of Mcm1 in vivo and in vitro, we have introduced an extensive series of base pair substitutions into an Mcm1 operator site and examined their effects on Mcm1-mediated transcriptional regulation and DNA-binding affinity. Our results show that Mcm1 uses a mechanism to contact the DNA that has some significant differences from the one used by the human serum response factor (SRF), a closely related MADS box protein in which the three-dimensional structure has been determined. One major difference is that 5-bromouracil-mediated photo-cross-linking experiments indicate that Mcm1 is in close proximity to functional groups in the major groove at the center of the recognition site whereas the SRF protein did not exhibit this characteristic. A more significant difference is that mutations at a position outside of the conserved CC(A/T)6GG site significantly reduce Mcm1-dependent DNA bending, while these substitutions have no effect on DNA bending by SRF. This result shows that the DNA bending by Mcm1 is sequence dependent and that the base-specific requirements for bending differ between Mcm1 and SRF. Interestingly, although these substitutions have a large effect on DNA bending and transcriptional activation by Mcm1, they have a relatively small effect on the DNA-binding affinity of the protein. This result suggests that the degree of DNA bending is important for transcriptional activation by Mcm1.
Rongo, C, Broihier HT, Moore L, Van Doren M, Forbes A, Lehmann R.  1997.  Germ plasm assembly and germ cell migration in Drosophila. Cold Spring Harb Symp Quant Biol. 62:1-11. AbstractWebsite
Many aspects of germ cell behavior, migration, and gonad formation are shared between vertebrate and invertebrate species. For example, a specialized germ plasm has been observed in many species including Caenorhabditis elegans and Xenopus. Furthermore, the fact that Vasa marks germ cells in many species suggests that even certain molecular aspects of germ cells may be common between different organisms. In most organisms, germ cells initially form at a location away from their target mesodermal tissues and have to migrate to reach the mesoderm. Further genetic studies will reveal the extent to which molecular aspects of germ cell migration and gonad formation are conserved.
Justice, MC, Hogan BP, Vershon AK.  1997.  Homeodomain-DNa Interactions of the Pho2 Protein are Promoter-dependent. Nucleic Acids Res. 25:4730-4739. Abstract
The homeodomain (HD) is a conserved sequence-specific DNA-binding motif found in many eukaryotic transcriptional regulatory proteins. Despite the wealth of in vitro data on the mechanism HD proteins use to bind DNA, comparatively little is known about the roles of individual residues in these domains in vivo . The Saccharomyces cerevisiae Pho2 protein contains a HD that shares significant sequence identity with the Drosophila Engrailed protein. We have used the co-crystal structure of Engrailed as a model to predict how Pho2 might contact DNA and have examined how individual residues of the Pho2 HD contribute to transcriptional activation in vivo and to DNA binding in vitro. Our results demonstrate that Pho2 and Engrailed share many similar DNA-binding characteristics. However, our results also show that some highly conserved residues, which contact the DNA in many HD structures, make relatively small contributions to the DNA-binding affinity and in vivo activity of the Pho2 protein. We also show that the N-terminal arm of the Pho2 HD is a critical component in determining the DNA-binding specificity of the protein and that the requirements for residues in the N-terminal arm are promoter-dependent for Pho2 transcriptional activation and DNA binding.