Secondary structure of the homeo domain of yeast alpha 2 repressor determined by NMR spectroscopy.

Phillips, Cynthia L.; Vershon, Andrew K.; Johnson, A D; Dahlquist, Frederick W.

doi:10.1101/gad.5.5.764

Cited by 56 publications

(48 citation statements)

References 33 publications

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“…The homeo domain is part of the carboxy-terminal domain, and NMR and crystallography experiments have shown that this region forms three a-helices that pack into a tight bundle (Phillips et al 1991;Wolberger et al 1991). A sketch of the a2 homeo domain bound to its DNA site is shown in Figure 1.…”

Section: Point Mutations In the Homeo Domain Of A2 Reduce The Level Omentioning

confidence: 99%

“…Homeo domain proteins have now been found in nearly all eukaryotic organisms, ranging from yeast to humans, and several hundred members of this DNA-binding family are known (for review, see Scott et al 1989;Hayashi and Scott 1990;Wuthrich and Gehring 1992}. The structures of five different homeo domains, including that of a2, have been determined by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography (Qian et al 1989;Kissinger et al 1990;Otting et al 1990;Phillips et al 1991;Wolberger et al 1991;Billeter et al 1993;Ceska et al 1993;Leiting et al 1993;Klemm et al 1994). Despite limited sequence identity among these homeo domains, they adopt nearly the same conformation.…”

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confidence: 99%

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A homeo domain protein lacking specific side chains of helix 3 can still bind DNA and direct transcriptional repression.

Vershon

Jin²,

Johnson³

1995

Genes Dev.

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A series of mutations in the homeo domain of the yeast a2 protein were constructed to test, both in vivo and in vitro, predictions based on the oL2-DNA cocrystal structure described by Wolberger et al. (1991). The effects of the mutations were observed in three different contexts using authentic target DNA sequences: a2 binding alone to specific DNA, ,v2 binding cooperatively with MCM1 to specific DNA, and a2 binding cooperatively with al to specific DNA. As expected, changes in the amino acid residues that contact DNA in the X-ray structure severely compromised the ability of a2 to bind DNA alone and to bind DNA cooperatively with MCM1. In contrast, many of these same mutations, including a triple change that altered all the "recognition" residues of helix 3, had little or no effect on the cooperative binding of a2 and al to specific DNA, as determined both in vivo and in vitro. These results show that the ability of a homeo domain protein to correctly select and repress target genes does not necessarily depend on the residues commonly implicated in sequence-specific DNA binding.

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Section: Point Mutations In the Homeo Domain Of A2 Reduce The Level Omentioning

confidence: 99%

mentioning

confidence: 99%

A homeo domain protein lacking specific side chains of helix 3 can still bind DNA and direct transcriptional repression.

Vershon

Jin²,

Johnson³

1995

Genes Dev.

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“…The nearly superimposable crystal structures of Engrailed and MAT α2 homeodomains show helix three almost at right angles to the DNA, and nestled in the major groove (Kissinger et al, 1990;Phillips et al, 1991;Wolberger et al, 1991). Four helix three residues (47, 50, 51 and 54) have an opportunity to contact the base pairs in the major groove.…”

Section: Specificity Determinants In Homeodomainsmentioning

confidence: 99%

“…In the MAT α2 structure, the long side chain of arginine 54 reaches back to make contact with the base pairs (Phillips et al, 1991), but in Engrailed this position is occupied by alanine whose short side chain does not participate in these interactions. Additionally, this position is not conserved among our sequences.…”

Section: Specificity Determinants In Homeodomainsmentioning

confidence: 99%

A universal target sequence is bound in vitro by diverse homeodomains

Kalionis

O’Farrell

1993

Mechanisms of Development

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To determine the number of DNA binding proteins capable of binding a consensus Engrailed binding site, this consensus sequence was used to screen a library of Drosophila cDNA clones in a bacteriophage expression vector. We retrieved clones encoding 20 distinct DNA binding domains, 17 of which are homeodomains. Binding to a variety of oligonucleotides confirms the related sequence specificity of the retrieved binding domains. Nonetheless, the homeodomains have remarkably diverse amino acid sequences. We conclude that during the evolutionary divergence of homeodomains, the specificity of DNA binding has been much more highly conserved than the amino acid sequence.

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“…Flexible regions adjacent to the N terminus and C terminus of the ␣2 homeodomain are required for interaction with the Mcm1 and a1 cofactors, respectively (26, 40 -44). The three-dimensional structure of the ␣2 homeodomain has been determined by NMR and x-ray crystallography studies (42,45,46). Although there is only 27% sequence identity between the ␣2 and Drosophila engrailed homeodomains, their overall structures are very similar (46,47).…”

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confidence: 99%

The Yeast Homeodomain Protein MATα2 Shows Extended DNA binding Specificity in Complex with Mcm1

Zhong

Vershon

1997

Journal of Biological Chemistry

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The MAT␣2 (␣2) repressor interacts with the Mcm1 protein to turn off a-cell type-specific genes in the yeast Saccharomyces cerevisiae. We compared five natural ␣2-Mcm1 sites with an ␣2-Mcm1 symmetric consensus site (AMSC) for their relative strength of repression and found that the AMSC functions slightly better than any of the natural sites. To further investigate the DNA binding specificity of ␣2 in complex with Mcm1, symmetric substitutions at each position in the ␣2 half-sites of AMSC were constructed and assayed for their effect on repression in vivo and DNA binding affinity in vitro. As expected, substitutions at positions in which there are base-specific contacts decrease the level of repression. Interestingly, substitutions at other positions, in which there are no apparent base-specific contacts made by the protein in the ␣2-DNA co-crystal structure, also significantly decrease repression. As an alternative method to examining the DNA binding specificity of ␣2, we performed in vitro ␣2 binding site selection experiments in the presence and absence of Mcm1. In the presence of Mcm1, the consensus sequences obtained were extended and more closely related to the natural ␣2 sites than the consensus sequence obtained in the absence of Mcm1. These results demonstrate that in the presence of Mcm1 the sequence specificity of ␣2 is extended to these positions.Homeodomain proteins are a family of transcription factors involved in many developmental and cellular processes and have been found in almost every eukaryotic organism (1-4). The natural target sites for many homeodomain proteins are unknown; therefore, their DNA-binding sites have been defined through site selection experiments in vitro (5-9). Although these studies provide important information on homeodomain-DNA recognition in vitro, in some cases it appears that the homeodomain proteins do not function well at these sites in vivo (10 -14). One possible explanation for this discrepancy is that in vivo the DNA binding specificity and affinity of some homeodomain proteins may be influenced by interactions with cofactors (13,(15)(16)(17)(18)(19). Since many of the studies which examine homeodomain binding sites have been done in the absence of cofactors, this may explain why in some cases sites selected in vitro may not be functional sites for the homeodomain protein complexes in vivo. To address this issue and to understand how homeodomain proteins recognize specific sites, we have investigated the DNA binding specificity of ␣2, a yeast homeodomain protein, in which the natural target sites and cofactors are well known.The ␣2 protein is involved in the regulatory system that specifies cell mating type in Saccharomyces cerevisiae (20 -24). In diploid cells, the ␣2 and a1 proteins form a heterodimer to repress expression of haploid-specific genes (25, 26). In haploid ␣ cells and diploid cells, ␣2 interacts with a general transcription regulatory factor, Mcm1, to repress expression of a-specific genes (asg) 1 (27-29). DNase I protection and deletion experimen...

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Secondary structure of the homeo domain of yeast alpha 2 repressor determined by NMR spectroscopy.

Cited by 56 publications

References 33 publications

A homeo domain protein lacking specific side chains of helix 3 can still bind DNA and direct transcriptional repression.

A homeo domain protein lacking specific side chains of helix 3 can still bind DNA and direct transcriptional repression.

A universal target sequence is bound in vitro by diverse homeodomains

The Yeast Homeodomain Protein MATα2 Shows Extended DNA binding Specificity in Complex with Mcm1

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