Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (322K) Freely available
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (11)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Moll, J. R.
Right arrow Articles by Vinson, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moll, J. R.
Right arrow Articles by Vinson, C.
Related Collections
Right arrow Protein-nucleic acid interaction
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2002, Vol. 30, No. 5 1240-1246
© 2002 Oxford University Press

Magnesium is required for specific DNA binding of the CREB B-ZIP domain

Jonathan R. Moll, Asha Acharya, Jozset Gal, Alain A. Mir and Charles Vinson*

Building 37, Room 4D06, Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

We have examined binding of the CREB B-ZIP protein domain to double-stranded DNA containing a consensus CRE sequence (5'-TGACGTCA-3'), the related PAR, C/EBP and AP-1 sequences and the unrelated SP1 sequence. DNA binding was assayed in the presence or absence of MgCl2 and/or KCl using two methods: circular dichroism (CD) spectroscopy and electrophoretic mobility shift assay (EMSA). The CD assay allows us to measure equilibrium binding in solution. Thermal denaturation in 150 mM KCl indicates that the CREB B-ZIP domain binds all the DNA sequences, with highest affinity for the CRE site, followed by the PAR (5'-TAACGTTA-3'), C/EBP (5'-TTGCGCAA-3') and AP-1 (5'-TGAGTCA-3') sites. The addition of 10 mM MgCl2 diminished DNA binding to the CRE and PAR DNA sequences and abolished binding to the C/EBP and AP-1 DNA sequences, resulting in more sequence-specific DNA binding. Using ‘standard’ EMSA conditions (0.25x TBE), CREB bound all the DNA sequences examined. The CREB–CRE complex had an apparent Kd of ~300 pM, PAR of ~1 nM, C/EBP and AP-1 of ~3 nM and SP1 of ~30 nM. The addition of 10 mM MgCl2 to the polyacrylamide gel dramatically altered sequence-specific DNA binding. CREB binding affinity for CRE DNA decreased 3-fold, but binding to the other DNA sequences decreased >1000-fold. In the EMSA, addition of 150 mM KCl to the gels had an effect similar to MgCl2. The magnesium concentration needed to prevent non-specific electrostatic interactions between CREB and DNA in solution is in the physiological range and thus changes in magnesium concentration may be a cellular signal that regulates gene expression.

* To whom correspondence should be addressed. Tel: +1 301 496 8753; Fax: +1 301 496 8419; Email: vinsonc{at}dc37a.nci.nih.gov


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Mol Biol EvolHome page
G. Amoutzias, A. Veron, J Weiner III, M Robinson-Rechavi, E Bornberg-Bauer, S. Oliver, and D. Robertson
One Billion Years of bZIP Transcription Factor Evolution: Conservation and Change in Dimerization and DNA-Binding Site Specificity
Mol. Biol. Evol., March 1, 2007; 24(3): 827 - 835.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
W. J. Oh, V. Rishi, A. Orosz, M. J. Gerdes, and C. Vinson
Inhibition of CCAAT/Enhancer Binding Protein Family DNA Binding in Mouse Epidermis Prevents and Regresses Papillomas
Cancer Res., February 15, 2007; 67(4): 1867 - 1876.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. J. Gerdes, M. Myakishev, N. A. Frost, V. Rishi, J. Moitra, A. Acharya, M. R. Levy, S.-w. Park, A. Glick, S. H. Yuspa, et al.
Activator protein-1 activity regulates epithelial tumor cell identity.
Cancer Res., August 1, 2006; 66(15): 7578 - 7588.
[Abstract] [Full Text] [PDF]

Home page
 Genome ResHome page
P. C. FitzGerald, A. Shlyakhtenko, A. A. Mir, and C. Vinson
Clustering of DNA Sequences in Human Promoters
Genome Res., August 1, 2004; 14(8): 1562 - 1574.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
V. Rishi, J. Gal, D. Krylov, J. Fridriksson, M. S. Boysen, S. Mandrup, and C. Vinson
SREBP-1 Dimerization Specificity Maps to Both the Helix-Loop-Helix and Leucine Zipper Domains: USE OF A DOMINANT NEGATIVE
J. Biol. Chem., March 19, 2004; 279(12): 11863 - 11874.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.