Sequence-selective DNA binding to the regulatory subunit of cAMP-dependent protein kinase.
The fluorescence of Trp-226 in the regulatory subunit of bovine type II cAMP-dependent protein kinase is unaffected by the binding of cAMP, but is quenched by the binding of 2'-dansyl-cAMP (DNS-cAMP). Up to 67% of the fluorescence of Trp-226 can be quenched by resonant energy transfer to the DNS-cAMP bound to the first site, and 96% of the fluorescence can be quenched by saturating both sites with DNS-cAMP. The observed efficiencies of energy transfer gave a distance of 16 A between Trp-226 and the DNS-cAMP bound at the first site and a distance of 12.7 A between Trp-226 and the DNS-cAMP bound at second site. The fluorescence of Trp-226 was suppressed by incubation of RII with the self-complementary octanucleotide TGACGTCA (CRE) due to binding of the oligonucleotide to RII. A detailed study of the binding equilibrium showed that each RII(cAMP)2 molecule binds 1 molecule of CRE with Kd = 80 nM. The corresponding Kd value for cAMP-depleted RII was found to be 25-fold higher. RII was also found to bind randomly selected DNA fragments with an average Kd value much higher than that of CRE. These observations show for the first time that the binding of oligonucleotide to RII is cAMP-enhanced and sequence-selective.
Wu
J C
JC
Bioenergetics Laboratory, State University of New York, Buffalo 14214.
Wang
J H
JH
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003619:Dansyl Compounds / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008024:Ligands; D008433:Mathematics; D009206:Myocardium / Q000201:enzymology; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D003619:Dansyl Compounds; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D009838:Oligodeoxyribonucleotides; C015482:protein kinase modulator; C059996:2'-dansyl-cyclic AMP; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases