Distinct phosphate backbone contacts revealed by some mutant peptides of zinc finger protein Spl: effect of protein-induced bending on DNA recognition.
By using some mutant peptides of transcription factor Spl, phosphate backbone contacts with the DNA binding protein containing three zinc fingers have been investigated by alkylation interference, circular permutation, DNase I footprinting, and methylation protection methods. The ethylation interference analyses of Spl(R565S) and Spl(K595S) mutants demonstrate that arginine at 565 position and lysine at 595 position interact with the phosphate between G(3) and G(4) and with the phosphate between G(9) and G(10) in GC-box DNA, respectively. On the basis of the experimental results for Spl(K535G), Sp1(537-623), and Sp1(530-623), lysine and glutamine at 535 and 536 positions have been clarified to be in contacts with phosphate between G(7) and G(8) and with phosphate outside GC-box, respectively. In particular, glutamine at the N-terminal side of zinc finger 1 is a key amino acid residue to induce DNA bending and also participates in total base specificity of Sp1. The present study strongly indicates that (1) each zinc finger is not independent for the DNA interaction with Sp1 and (2) DNA base recognition of the zinc finger protein is influenced by local conformational change of DNA induced by the protein binding.
Nagaoka
M
M
Institute for Chemical Research, Kyoto University, Japan.
Sugiura
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000737:chemistry; D008745:Methylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010710:Phosphates / Q000737:chemistry*; D016329:Sp1 Transcription Factor / Q000737:chemistry*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D010710:Phosphates; D016329:Sp1 Transcription Factor; D004247:DNA; D003850:Deoxyribonuclease I