Asymmetrical recognition of the palindromic AP1 binding site (TRE) by Fos protein complexes.
Fos and Jun proteins form a tight complex which binds specifically to the AP1 recognition sequence, a palindromic DNA element also referred to as the TPA responsive element (TRE). To elucidate the mechanism of Fos-Jun interaction with the TRE we have performed UV cross-linking studies using oligonucleotides where thymines were replaced with bromouracil. Our results indicate that both Fos and Jun directly contact the TRE but that the interaction of Fos and Jun with thymines in structurally equivalent positions in the two half sites of the TRE is different. In addition, we have carried out a comprehensive mutagenesis study of the TRE by introducing all possible point mutations plus thymine----uracil substitutions into the palindromic TRE core sequences and the adjacent nucleotides on both sides. The results of this analysis clearly show that the palindromic TRE is asymmetrical with respect to binding of Fos-Jun. We also show that a Fos protein complex with a homodimeric DNA binding site binds considerably less efficiently to TRE mutants with a perfect dyad symmetry compared with the binding to the wild-type TRE. This demonstrates that the asymmetrical recognition of the TRE is not due to the heterodimeric nature of the Fos/Jun complex but directly related to an asymmetry in the TRE sequence. The methyl groups of all four thymine residues within the TRE seem to be functionally crucial since thymine----uracil substitutions strongly reduce or abolish binding to Fos/Jun. The relevance of structurally equivalent methyl groups in the TRE core sequence is different, lending further support to the conclusion that the TRE is asymmetrical.
Risse
G
G
Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, FRG.
Jooss
K
K
Neuberg
M
M
Brüller
H J
HJ
Müller
R
R
D000818:Animals; D001483:Base Sequence / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D013755:Tetradecanoylphorbol Acetate