title: C-terminal in Sp1-like artificial zinc-finger proteins plays crucial roles in determining their DNA binding affinity.
abstract: BACKGROUND
  It is well known that the C-terminal zinc-finger-3 in transcription factor Sp1 contributes more than the N-terminal zinc-finger-1 in determining Sp1's DNA binding capacity. Sp1-like artificial poly-zinc-finger proteins (ZFPs) are powerful biotechnological tools for gene-specific recognization and manipulation. It is important to understand whether the C-terminal fingers in the Sp1-like artificial ZFPs remain crucial for their DNA binding ability. Recently, a set of p16 promoter-specific seven-ZFPs (7ZFPs) has been constructed to reactivate the expression of methylation-silenced p16. These 7ZFPs contain one N-terminal three-zinc-finger domain of Sp1 (3ZF), two Sp1-like two-zinc-finger domains derived from the Sp1 finger-2 and finger-3 (2ZF) in the middle and C-terminal regions.
  
  
  RESULTS
  In the present study, sets of variants for several representative 7ZFPs with the p16-binding affinity were further constructed. This was accomplished through finger replacements and key amino acid mutations in the N-terminal fingers, C-terminal fingers, and linker peptide, respectively. Their p16-binding activity was analysed using gel mobility shift assays. Results showed that the motif replacement or a key amino acid mutation (S > R) at position +2 of the α-helix in the C-terminal 2ZF domain completely abolished their p16-binding affinity. Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity. In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability.
  
  
  CONCLUSIONS
  Altogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.
authors.forename: Baozhen
authors.initials: B
authors.lastname: Zhang
authors.forename: Shengyan
authors.initials: S
authors.lastname: Xiang
authors.forename: Yanru
authors.initials: Y
authors.lastname: Yin
authors.forename: Liankun
authors.initials: L
authors.lastname: Gu
authors.affiliation: Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China. dengdajun@bjmu.edu.cn.
authors.forename: Dajun
authors.initials: D
authors.lastname: Deng
mesh_terms: D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D016329:Sp1 Transcription Factor / Q000737:chemistry* / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D016329:Sp1 Transcription Factor