Involvement of lysine residues 289 and 291 of the cAMP-responsive element-binding protein in the recognition of the cAMP-responsive element.
The molecular interactions resulting in specific binding of trans-acting factors to distinct cis-acting elements is not well understood. Here we report our attempt to understand the involvement of distinct amino acid residues of the basic domain of cAMP-responsive element-binding protein (delta CREB) in the determination of binding toward the cAMP-responsive element (CRE). Using in vitro mutagenesis, we constructed site-directed mutants of distinct amino acid residues within the DNA contact region of delta CREB. The activities of the mutant proteins were analyzed by gel retardation, methylation interference, and CRE competition analyses. We demonstrate that a single lysine to glutamine substitution at positions 289 and 291 of delta CREB alters the methylation interference pattern of the mutant protein for the CRE site. Additional mutants constructed at these positions demonstrate that only identical basic residues at both positions 289 and 291 of delta CREB can restore the wild type methylation interference pattern of the mutant delta CREB protein for the CRE site. These observations point to the importance of the lysine residues at positions 289 and 291 in the process of CRE binding. In addition, this observation suggests that the symmetrical nature of the CRE site is reflected in the DNA contact region of the protein.
Andrisani
O M
OM
Department of Veterinary Physiology and Pharmacology, Purdue University, West Lafayette, Indiana 47907.
Dixon
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007231:Infant, Newborn; D016350:Leucine Zippers / Q000235:genetics*; D008239:Lysine; D046911:Macromolecular Substances; D008745:Methylation; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D017362:Cyclic AMP Response Element-Binding Protein; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D008239:Lysine