GFP-linked zinc finger protein sp1. fluorescence study and implication for N-terminal zinc finger 1 as hinge finger.
The N-terminal zinc finger (zf) of Sp1 is referred to as the 'hinge finger', which connects the C-terminal DNA binding domain with the N-terminal activation domain. In this study, we investigated how a green fluorescent protein (GFP) linked to the N-terminal zinc finger is located spatially. The fluorescence resonance energy transfer technique and steady-state fluorescence anisotropy measurements indicate the results as follows: (1) In the binding to GC-box DNA, the geometry of the GFP domain of the GFP-linked Sp1 zinc finger is similar to that of the Ala-556-->Arg mutant. (2) The GFP-linked Sp1 zinc finger is folded more compactly in the absence of DNA (hydrodynamic volume V=78.2 nm(3)) and consequently alters the conformation at the GFP domain more extensively (DeltaV=43.6 nm(3)) upon DNA binding than the Ala-556-->Arg mutant (99.5, 14.8 nm(3), respectively). These results implicate that the N-terminal 'hinge finger' moderates various interactions of the adjacent N-terminal regulation domain with other transcriptional factors as well as DNA binding and is essential for the function of Sp1 and/or Sp/XKLF family members.
Matsushita
Keizo
K
Institute for Chemical Research, Kyoto University, Uji, Japan.
Sugiura
Yukio
Y
D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D024202:Electrophoretic Mobility Shift Assay; D005454:Fluorescence Polarization / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D020862:GC Rich Sequence; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016329:Sp1 Transcription Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
D008164:Luminescent Proteins; D009841:Oligonucleotides; D016329:Sp1 Transcription Factor; D049452:Green Fluorescent Proteins; D004247:DNA; D005982:Glutathione Transferase