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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.

Right-side-out vesicles of pig kidney microsomes and amino-acid-sequence-specific antibodies were used to probe the sidedness of the C-terminus and the N-terminus of the catalytic CI subunit of Na+/K+-ATPase. Polyclonal antibodies were raised in rabbits against the peptide corresponding to the N-terminal sequence GRDKYEPAAVSE (peptide 1 -12) and against peptides corresponding to the C-terminal sequences IFVYDEVRKLIIRRR (peptide 991 -1005) and RPGGWVEKETYY (peptide 1005-1016). These antibodies were purified by affinity chromatography on the respective peptide-Sepharose columns. Moreover, antibodies against the N-terminal dodecapeptide GRDKYEPAAVSE were obtained by affinity purification from heteroclonal antibodies against the CI subunit of pork kidney Na+/K+-ATPase. These antibodies reacted with native as well as SDS-denaturated Na+/K+-ATPase. When the antibodies were used to probe the sidedness of the sequences in right-side-out vesicles of pig kidney microsomes, the N-terminal peptide 1 -12 as well as the C-terminal peptides 991 -1005 and 1005 -101 6 were found on the cytosolic side. Concanavalin A, however, which interacts with the j3 subunit, a glycoprotein, reacted with the outside of right-side-out vesicles.

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