Format

Send to

Choose Destination
Biochemistry. 2012 Dec 18;51(50):10024-34. doi: 10.1021/bi301306t. Epub 2012 Dec 10.

Identification of natural and artificial DNA substrates for light-activated LOV-HTH transcription factor EL222.

Author information

1
Departments of Biophysics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-8816, USA.

Abstract

Light-oxygen-voltage (LOV) domains serve as the photosensory modules for a wide range of plant and bacterial proteins, conferring blue light-dependent regulation to effector activities as diverse as enzymes and DNA binding. LOV domains can also be engineered into a variety of exogenous targets, allowing similar regulation for new protein-based reagents. Common to these proteins is the ability for LOV domains to reversibly form a photochemical adduct between an internal flavin chromophore and the surrounding protein, using this to trigger conformational changes that affect output activity. Using the Erythrobacter litoralis protein EL222 model system that links LOV regulation to a helix-turn-helix (HTH) DNA binding domain, we demonstrated that the LOV domain binds and inhibits the HTH domain in the dark, releasing these interactions upon illumination [Nash, A. I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 9449-9454]. Here we combine genomic and in vitro selection approaches to identify optimal DNA binding sites for EL222. Within the bacterial host, we observe binding at several genomic sites using a 12 bp sequence consensus that is also found by in vitro selection methods. Sequence-specific alterations in the DNA consensus reduce EL222 binding affinity in a manner consistent with the expected binding mode, a protein dimer binding to two repeats. Finally, we demonstrate the light-dependent activation of transcription of two genes adjacent to an EL222 binding site. Taken together, these results shed light on the native function of EL222 and provide useful reagents for further basic and applications research of this versatile protein.

PMID:
23205774
PMCID:
PMC3531242
DOI:
10.1021/bi301306t
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for American Chemical Society Icon for PubMed Central
Loading ...
Support Center