Magnesium is required for specific DNA binding of the CREB B-ZIP domain.
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 approximately 300 pM, PAR of approximately 1 nM, C/EBP and AP-1 of approximately 3 nM and SP1 of approximately 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.
Moll
Jonathan R
JR
Building 37, Room 4D06, Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Acharya
Asha
A
Gal
Jozsef
J
Mir
Alain A
AA
Vinson
Charles
C
Gal
Jozset
J
D000818:Animals; D030161:Avian Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002942:Circular Dichroism; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D008274:Magnesium / Q000494:pharmacology / Q000502:physiology*; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013696:Temperature; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D030161:Avian Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D004247:DNA; D008274:Magnesium