Structural Basis for Dimerization in DNA Recognition by Gal4

Hong, Manqing; Fitzgerald, Mary X.; Harper, Sandra L.; Luo, Cheng; Speicher, David W.; Marmorstein, Ronen

doi:10.1016/j.str.2008.03.015

Cited by 79 publications

(78 citation statements)

References 38 publications

(41 reference statements)

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“…Each DBD-TAD fusion protein exhibited an identical binding affinity (K d ϭ 15 Ϯ 5 nM) for a fluorescently labeled consensus DNA-binding site composed of two half-sites as determined by fluorescence polarization experiments (supplemental Fig. S3); these K d values are consistent with those previously reported for Gal4(1-100) (31). Thus, the DNA-binding function was independent of the TAD, indicative of the modular architecture of most transcriptional activators (5,6).…”

Section: Dna-binding Propertiessupporting

confidence: 88%

Transient-state Kinetic Analysis of Transcriptional Activator·DNA Complexes Interacting with a Key Coactivator

Wands¹,

Wang

Lum

et al. 2011

Journal of Biological Chemistry

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Several lines of evidence suggest that the prototypical amphipathic transcriptional activators Gal4, Gcn4, and VP16 interact with the key coactivator Med15 (Gal11) during transcription initiation despite little sequence homology. Recent cross-linking data further reveal that at least two of the activators utilize the same binding surface within Med15 for transcriptional activation. To determine whether these three activators use a shared binding mechanism for Med15 recruitment, we characterized the thermodynamics and kinetics of Med15⅐activator⅐DNA complex formation by fluorescence titration and stopped-flow techniques. Combination of each activator⅐DNA complex with Med15 produced biphasic time courses. This is consistent with a minimum twostep binding mechanism composed of a bimolecular association step limited by diffusion, followed by a conformational change in the Med15⅐activator⅐DNA complex. Furthermore, the equilibrium constant for the conformational change (K 2 ) correlates with the ability of an activator to stimulate transcription. VP16, the most potent of the activators, has the largest K 2 value, whereas Gcn4, the least potent, has the smallest value. This correlation is consistent with a model in which transcriptional activation is regulated at least in part by the rearrangement of the Med15⅐activator⅐DNA ternary complex. These results are the first detailed kinetic characterization of the transcriptional activation machinery and provide a framework for the future design of potent transcriptional activators.

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Section: Dna-binding Propertiessupporting

confidence: 88%

Transient-state Kinetic Analysis of Transcriptional Activator·DNA Complexes Interacting with a Key Coactivator

Wands¹,

Wang

Lum

et al. 2011

Journal of Biological Chemistry

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“…GAL4(148) binds DNA as a homodimer, and deletion of its dimerization domain (66–100 amino acids) abolishes this binding 28 . Indeed, the truncated GAL4(68) fused with VP16 failed to induce EGFP reporter expression, in contrast to GAL4(148)–VP16 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Near-infrared optogenetic pair for protein regulation and spectral multiplexing

et al. 2017

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Multifunctional optogenetic systems are in high demand for use in basic and biomedical research. Near-infrared-light-inducible binding of bacterial phytochrome BphP1 to its natural PpsR2 partner is beneficial for simultaneous use with blue-light-activatable tools. However, applications of the BphP1–PpsR2 pair are limited by the large size, multidomain structure and oligomeric behavior of PpsR2. Here, we engineered a single-domain BphP1 binding partner, Q-PAS1, which is three-fold smaller and lacks oligomerization. We exploited a helix–PAS fold of Q-PAS1 to develop several near-infrared-light-controllable transcription regulation systems, enabling either 40-fold activation or inhibition. The light-induced BphP1–Q-PAS1 interaction allowed modification of the chromatin epigenetic state. Multiplexing the BphP1–Q-PAS1 pair with a blue-light-activatable LOV-domain-based system demonstrated their negligible spectral crosstalk. By integrating the Q-PAS1 and LOV domains in a single optogenetic tool, we achieved tridirectional protein targeting, independently controlled by near-infrared and blue light, thus demonstrating the superiority of Q-PAS1 for spectral multiplexing and engineering of multicomponent systems.

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“…In the presence of cellulose, the most highly expressed TF gene in the Dcre1 strain, relative to expression in strain QM9414, encodes a Zn2Cys6-type transcriptional regulator (ID 121682). The function of this gene has not yet been described, but the encoded protein contains a Gal4 domain, which is involved in galactose and melibiose metabolism in yeast (Hong et al, 2008). Another gene regulated by CRE1 in cellulose encodes the bZIP transcriptional regulator MeaB (ID 73417).…”

Section: Expression Of Transcription Factors Genesmentioning

confidence: 99%