Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space

Many genomes contain families of paralogs—proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD’s ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors’ response element-binding specificity were far from the proteins’ DNA-binding interface and interacted epistatically to change the DBD’s function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.

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Correlated Evolutionary Pressure at Interacting Transcription Factors and DNA Response Elements Can Guide the Rational Engineering of DNA Binding Specificity

Journal of Molecular Biology ◽

10.1016/j.jmb.2005.04.054 ◽

2005 ◽

Vol 350 (3) ◽

pp. 402-415 ◽

Cited By ~ 21

Author(s):

Michele Raviscioni ◽

Peili Gu ◽

Minawar Sattar ◽

Austin J. Cooney ◽

Olivier Lichtarge

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Binding Specificity ◽

Response Elements ◽

Rational Engineering ◽

Dna Binding Specificity

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A single amino-acid substitution in the Ets domain alters core DNA binding specificity of Ets1 to that of the related transcription factors Elf1 and E74

Nucleic Acids Research ◽

10.1093/nar/21.22.5184 ◽

1993 ◽

Vol 21 (22) ◽

pp. 5184-5191 ◽

Cited By ~ 35

Author(s):

Rémy Bosselut ◽

Jonathan Levin ◽

Elisabeth Adjadj ◽

Jacques Ghysdael

Keyword(s):

Transcription Factors ◽

Amino Acid ◽

Dna Binding ◽

Amino Acid Substitution ◽

Binding Specificity ◽

Single Amino Acid Substitution ◽

Single Amino Acid ◽

Dna Binding Specificity ◽

Ets Domain

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DNA-binding specificity of GATA family transcription factors.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3999 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3999-4010 ◽

Cited By ~ 448

Author(s):

M Merika ◽

S H Orkin

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Mammalian Cells ◽

Chimeric Protein ◽

Binding Specificity ◽

Mobility Shift ◽

Binding Domains ◽

Dna Binding Specificity ◽

Mobility Shift Assay ◽

Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

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The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5128 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5128-5137 ◽

Cited By ~ 10

Author(s):

M M Witte ◽

R C Dickson

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Zinc Finger ◽

Binding Specificity ◽

Binding Domain ◽

Dna Binding Domain ◽

Transcription Activator ◽

Activator Proteins ◽

Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

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Switching DNA-binding specificity by unnatural amino acid substitution

Nucleic Acids Research ◽

10.1093/nar/gki899 ◽

2005 ◽

Vol 33 (18) ◽

pp. 5896-5903 ◽

Cited By ~ 4

Author(s):

A. Maiti

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Amino Acid Substitution ◽

Binding Specificity ◽

Unnatural Amino Acid ◽

Dna Binding Specificity

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Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3850-3859.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3850-3859

Author(s):

T A Coleman ◽

C Kunsch ◽

M Maher ◽

S M Ruben ◽

C A Rosen

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Fusion Protein ◽

Binding Specificity ◽

Single Amino Acid ◽

Nf Kappa B ◽

Dna Binding Specificity ◽

Dna Motif ◽

Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

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