Crosslinking transcription factors to their recognition sequences with PtII complexes.
We have prepared phosphorothioate-containing cyclic oligodeoxynucleotides that fold into 'dumbbells' containing CRE and TRE sequences, the binding sequences for the CREB and JUN proteins, respectively. Six phosphorothioate residues were introduced into each of the recognition sequences. K2PtCl4 crosslinks CRE to CREB and TRE to JUN. The extent of crosslinking is about eight times greater than that observed with standard oligodeoxynucleotides and amounts to 30-50% of the efficiency of non-covalent association as estimated by gel-shift assays. Crosslinking is reversed by incubation with NaCN. The crosslinking reaction is specific--a dumbbell oligonucleotide with six phosphorothioate groups introduced into the Sp1 recognition sequence could not be crosslinked efficiently to CREB or JUN proteins with K2PtCl4. The binding of TRE to CREB is not strong enough for effective detection by gel-shift assays, but the TRE-CREB complex is crosslinked efficiently by K2PtCl4 and can then readily be detected.
Chu
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Salk Institute for Biological Studies, San Diego, CA 92186-5800.
Orgel
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LE
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002712:Chlorides / Q000737:chemistry*; D003432:Cross-Linking Reagents; D017362:Cyclic AMP Response Element-Binding Protein; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D010984:Platinum / Q000737:chemistry*; D017671:Platinum Compounds; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry* / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D002712:Chlorides; D003432:Cross-Linking Reagents; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D017671:Platinum Compounds; D016755:Proto-Oncogene Proteins c-jun; D013873:Thionucleotides; D014157:Transcription Factors; C074084:nuclear factor-jun; C036919:tetrachloroplatinate; D010984:Platinum; D004247:DNA