DNA-BINDING SPECIFICITIES AND PAIRING RULES OF THE AH RECEPTOR, ARNT,AND SIM PROTEINS

The Ah receptor (AHR), the Ah receptor nuclear translocator protein (A RNT), and single-minded protein (SIM) are members of the basic helix-l oop-helix-PAS (bHLH-PAS) family of regulatory proteins, In this study, we examine the DNA half-site recognition and pairing rules for these proteins using oligonucleotide selection-amplification and coprecipita tion protocols, Oligonucleotide selection-amplification revealed that a variety of bHLH-PAS protein combinations could interact, with each g enerating a unique DNA binding specificity. To validate the selection- amplification protocol, we demonstrated the preference of the AHR ARNT complex for the sequence commonly found in dioxin-responsive enhancer s in vivo (TNGCGTG). We then demonstrated that the ARNT protein is cap able of forming a homodimer with a binding preference for the palindro mic E-box sequence, CACGTG. Further examination indicated that ARNT ma y have a relaxed partner specificity, since it was also capable of for ming a heterodimer with SIM and recognizing the sequence GT(G/A)CGTG, Coprecipitation experiments using various PAS proteins and ARNT were c onsistent with the idea that the ARNT protein has a broad range of int eractions among the bHLH-PAS proteins, while the other members appear more restricted in their interactions, Comparison of this in vibro dat a with sites known to be bound in vivo suggests that the high affinity half-site recognition sequences for the AHR, SIM, and ARNT are T(C/T) GC, GT(G/A)C (5'-half-sites), and GTG (3'-half-sites), respectively.