Significant effect of linker sequence on DNA recognition by multi-zinc finger protein.
The unique linker sequence of the native nine zinc finger transcription factor IIIA (TFIIIA) appears to significantly affect its novel DNA recognition mode. An artificial new nine zinc finger peptide Sp1ZF9T has been created by connecting three units of the three zinc finger domains of Sp1 with the TFIIIA-type linker. The DNA-binding characteristics of Sp1ZF9T were evaluated by the gel mobility shift, DNase I footprinting, and methylation interference assays, and compared with those of the previous Sp1ZF9 with a Krüppel-type linker. The gel mobility shift assays revealed that Sp1ZF9T forms two complex species, a short-lived species (B-2) and a long-lived species (B-1), with GCIII DNA (5'-GGG GCG GGG GGG GCG GGG GGG GCG GGGCC-3'). The B-2 complex dissociated into the free peptide and DNA, whereas the B-1 complex was stable even after 72 h. The DNase I footprinting and methylation interference results indicated that 3'- and central portions of GCIII DNA are recognized by Sp1ZF9T in the B-1 complex. The present DNA binding mode of Sp1ZF9T is evidently different from that of Sp1ZF9. Namely, fingers 1-5 participate in the DNA contact of Sp1ZF9T, and fingers 1-9 in that of Sp1ZF9. Therefore, the linker sequence among the zinc finger domains has a significant effect on the specific DNA recognition by the multi-zinc finger proteins.
Nagaoka
M
M
Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011, Japan.
Nomura
W
W
Shiraishi
Y
Y
Sugiura
Y
Y
D000818:Animals; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000737:chemistry; D020862:GC Rich Sequence; D051741:Kruppel-Like Transcription Factors; D008954:Models, Biological; D010455:Peptides / Q000378:metabolism; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000378:metabolism*; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000737:chemistry*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D010455:Peptides; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D003850:Deoxyribonuclease I