Only one of the two DNA-bound orientations of AP-1 found in solution cooperates with NFATp.
BACKGROUND
The transcription factor AP-1 activates the expression of numerous genes in response to mitogenic stimuli. AP-1 regulates gene expression both through solitary binding to independent recognition sites and, in cooperation with various heterologous transcription factors, through targeting to composite response elements. The two subunits that make up the AP-1 heterodimer, Fos and Jun, possess identical residues at positions that make sequence-specific contacts to DNA. This degeneracy leaves the protein with no apparent way of orienting itself uniquely on DNA by differentially recognizing its two non-identical half-sites. Here, we have analyzed the orientation of the AP-1 basic-leucine-zipper (bZip) domain on a cognate site, both alone and in the cooperative complex formed together with the 'nuclear factor of activated T cells' (NFATp).


RESULTS
The results of affinity cleaving experiments demonstrate that, in solution, the AP-1 bZip binds DNA as a mixture of two orientational isomers. However, in the cooperative complex formed with NFATp on a composite response element, the AP-1 bZip adopts a single orientation, with Jun and Fos bound to the NFATp-proximal and NFATp-distal half-sites, respectively. Protein cross-linking experiments demonstrate that protein-protein contacts are responsible for this 'orientational locking'.


CONCLUSIONS
Our results demonstrate that, through protein-protein interactions, one protein can force another to adopt a single DNA-bound orientation. Thus, cooperative interactions between adjacent regulatory proteins can influence not only the energetics of their interactions with DNA, but also their precise geometric and stereochemical arrangement. Because orientational isomers present markedly different structures to the transcriptional apparatus, it seems likely that orientation will exert an effect on the ability to activate transcription.
Chen
L
L
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138, USA.
Oakley
M G
MG
Glover
J N
JN
Jain
J
J
Dervan
P B
PB
Hogan
P G
PG
Rao
A
A
Verdine
G L
GL
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D004247:DNA