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

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

Research output: Contribution to journalReview articlepeer-review

53 Scopus citations

Abstract

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 MgCI2 and/or KCI 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 KCI 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 MgCI2 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 (0.25× 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 MgCI2 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 KCI to the gels had an effect similar to MgCI2. 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.

Original languageEnglish
Pages (from-to)1240-1246
Number of pages7
JournalNucleic Acids Research
Volume30
Issue number5
DOIs
StatePublished - Mar 1 2002

ASJC Scopus subject areas

  • Genetics

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