DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins.
The Ah receptor (AHR), the Ah receptor nuclear translocator protein (ARNT), and single-minded protein (SIM) are members of the basic helix-loop-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 coprecipitation protocols. Oligonucleotide selection-amplification revealed that a variety of bHLH-PAS protein combinations could interact, with each generating 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 enhancers in vivo (TNGCGTG). We then demonstrated that the ARNT protein is capable of forming a homodimer with a binding preference for the palindromic E-box sequence, CACGTG. Further examination indicated that ARNT may have a relaxed partner specificity, since it was also capable of forming a heterodimer with SIM and recognizing the sequence GT(G/A)CGTG. Coprecipitation experiments using various PAS proteins and ARNT were consistent with the idea that the ARNT protein has a broad range of interactions among the bHLH-PAS proteins, while the other members appear more restricted in their interactions. Comparison of this in vitro data 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.
Swanson
H I
HI
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Chan
W K
WK
Bradfield
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D002474:Cell-Free System; D016384:Consensus Sequence; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D029721:Drosophila Proteins; D005786:Gene Expression Regulation; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D014176:Protein Biosynthesis; D011817:Rabbits; D018336:Receptors, Aryl Hydrocarbon / Q000096:biosynthesis / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; C089449:sim protein, Drosophila