Finger-positional change in three zinc finger protein Sp1: influence of terminal finger in DNA recognition.
The connection of functional modules is effective for the design of DNA binding molecules with the desired sequence specificity. C(2)H(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules and are expected to recognize any DNA sequences by permutation, multi-connection, and the substitution of various sets of zinc fingers. To investigate the effects of the replacement of the terminal finger on the DNA recognition by other fingers, we have constructed the three zinc finger peptides with finger substitution at the N- or C-terminus, Sp1(zf223), Sp1(zf323), and Sp1(zf321). From the results of gel mobility shift assays, each mutant peptide binds preferentially to the target sequence that is predicted if the fingers act in a modular fashion. The methylation interference analyses demonstrate that in the cases of the N-terminal finger substitution mutants, Sp1(zf223) and Sp1(zf323), the N-terminal finger recognizes bases to different extents from that of the wild-type peptide, Sp1(zf123). Of special interest is the fact that the N-terminal finger of the C-terminal finger substitution mutant, Sp1(zf321), shows a distinct base recognition from those of Sp1(zf123) and Sp1(zf323). DNase I footprinting analyses indicate that the C-terminal finger (active finger) induces a conformational change in the DNA in the region for the binding of the N-terminal finger (passive finger). The present results strongly suggest that the extent of base recognition of the N-terminal finger is dominated by the binding of the C-terminal finger. This information provides an important clue for the creation of a zinc finger peptide with the desired specificity, which is applicable to the design of novel drugs and biological tools.
Uno
Y
Y
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Matsushita
K
K
Nagaoka
M
M
Sugiura
Y
Y
D001482:Base Composition; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D006801:Humans; D009690:Nucleic Acid Conformation; D009842:Oligopeptides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D017510:Protein Folding; D016329:Sp1 Transcription Factor / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D016335:Zinc Fingers / Q000235:genetics
D009842:Oligopeptides; D016329:Sp1 Transcription Factor; D004247:DNA