Characterization of the DNA binding properties of the bHLH domain of Deadpan to single and tandem sites

被引：26

作者：

Winston, RL ^{[1
]}

Millar, DP ^{[1
]}

Gottesfeld, JM ^{[1
]}

Kent, SBH ^{[1
]}

机构：

[1] Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA

来源：

BIOCHEMISTRY | 1999年 / 38卷 / 16期

关键词：

D O I：

10.1021/bi982856a

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The basic helix-loop-helix domain of the Drosophila transcription factor Deadpan (Dpn) was prepared by total chemical protein synthesis in order to characterize its DNA binding properties. Circular dichroism spectroscopy was used to correlate structural changes in Dpn with physiologically relevant monovalent (KCI) and divalent (MgCl2) cation concentrations. In addition, we have used electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy methods to determine equilibrium dissociation constants for the interaction of Dpn with two biologically relevant promoters involved in neural development and sex determination pathways. In this study, we have optimized DNA binding conditions for Dpn, and we have found a markedly higher DNA binding affinity for Dpn than reported for other bHLH domain transcription factors. Dpn binds as a homodimer (K-d = 2.6 nM) to double-stranded oligonucleotides containing the binding site CACGCG. In addition, we found that Dpn bound with the same affinity to a single or tandem binding site, indicating no cooperativity between adjacent DNA-bound Dpn dimers. DNA binding was also monitored as a function of physiologically relevant KCI and MgCl2 concentrations, and we found that this activity was significantly different in the presence and absence of the nonspecific competitor poly(dI-dC). Moreover, Dpn displayed moderate sequence selectivity, exhibiting a 100-fold higher binding affinity for specific DNA than for poly(dI-dC). This study constitutes the first detailed biophysical characterization of the DNA binding properties of a bHLH protein.

引用

页码：5138 / 5146

页数：9

共 49 条

[1]

AKAZAWA C, 1992, J BIOL CHEM, V267, P21879

[2] MOLECULAR CHARACTERIZATION OF HELIX-LOOP-HELIX PEPTIDES [J].

ANTHONYCAHILL, SJ ;

BENFIELD, PA ;

FAIRMAN, R ;

WASSERMAN, ZR ;

BRENNER, SL ;

STAFFORD, WF ;

ALTENBACH, C ;

HUBBELL, WL ;

DEGRADO, WF .

SCIENCE, 1992, 255 (5047) :979-983

[3] A natural classification of the basic helix-loop-helix class of transcription factors [J].

Atchley, WR ;

Fitch, WM .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1997, 94 (10) :5172-5176

[4] Hairy mediates dominant repression in the Drosophila embryo [J].

Barolo, S ;

Levine, M .

EMBO JOURNAL, 1997, 16 (10) :2883-2891

[5] DEADPAN, AN ESSENTIAL PAN NEURAL GENE IN DROSOPHILA, ENCODES A HELIX LOOP HELIX PROTEIN SIMILAR TO THE HAIRY GENE-PRODUCT [J].

BIER, E ;

VAESSIN, H ;

YOUNGERSHEPHERD, S ;

JAN, LY ;

JAN, YN .

GENES & DEVELOPMENT, 1992, 6 (11) :2137-2151

[6] Conservation of the Drosophila lateral inhibition pathway in human lung cancer: A hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression [J].

Chen, H ;

Thiagalingam, A ;

Chopra, H ;

Borges, MW ;

Feder, JN ;

Nelkin, BD ;

Baylin, SB ;

Ball, DW .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1997, 94 (10) :5355-5360

[7] DETERMINATION OF HELIX AND BETA-FORM OF PROTEINS IN AQUEOUS-SOLUTION BY CIRCULAR-DICHROISM [J].

CHEN, YH ;

YANG, JT ;

CHAU, KH .

BIOCHEMISTRY, 1974, 13 (16) :3350-3359

[8] THE MYOD DNA-BINDING DOMAIN CONTAINS A RECOGNITION CODE FOR MUSCLE-SPECIFIC GENE ACTIVATION [J].

DAVIS, RL ;

CHENG, PF ;

LASSAR, AB ;

WEINTRAUB, H .

CELL, 1990, 60 (05) :733-746

[9]

DAWSON SR, 1995, MOL CELL BIOL, V15, P6923

[10] THE ENHANCER OF SPLIT [E(SPL)] LOCUS OF DROSOPHILA ENCODES 7 INDEPENDENT HELIX LOOP HELIX PROTEINS [J].

DELIDAKIS, C ;

ARTAVANISTSAKONAS, S .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (18) :8731-8735

← 1 2 3 4 5 →