一种特异识别SV40启动子的人工转录因子的构建

转录因子是真核表达调控中非常重要的一类反式作用因子,通常由DNA结合域与效应域两部分组成,而锌指结构是DNA结合域的常见组成单元。人工转录因子就是基于转录因子的这种结构特点,人为地选择针对特定序列的DNA结合域与具有特定作用的效应域组合而成。利用噬菌体展示技术,筛选到与SV40启动子上9bp序列特异结合的锌指结构,再连接KOX1的KRAB域构建了一种人工转录因子。转染实验表明它对SV40下游的报告基因的表达有很显著的抑制作用。     

Validated zinc finger protein designs for all 16 GNN DNA triplet targets.

The Cys(2)-His(2)-type zinc finger DNA-binding proteins can be engineered to bind specifically to many different DNA sequences. A single zinc finger typically binds to a 3-4-base pair DNA subsite. One strategy for design is to identify highly specific fingers that recognize each of the 64 possible DNA triplets. We started with a subgroup of the 64 triplets, the GNN-binding fingers. The GNN-binding fingers have been examined in several studies, but previous studies did not produce specific fingers for all of the 16 GNN triplets. These previous studies did not provide any information on the possible positional or context effects on the performance of these fingers. To identify the most specific design and take the possible positional effects into consideration, we did a large-scale site selection experiment on our GNN designs. From this study, we identified very specific fingers for 14 of the 16 GNN triplets, demonstrating for the first time a clear positional dependence for many of the designs. Further systematic specificity study reveals that the in vivo functionality of these zinc finger proteins in a reporter assay depends on their binding affinities to their target sequences, thus giving a better understanding of how these zinc finger proteins might function inside cells.     

Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d

The tandem zinc finger (TZF) domain of the protein TIS11d binds to the class II AU-rich element (ARE) in the 3' untranslated region (3' UTR) of target mRNAs and promotes their deadenylation and degradation. The NMR structure of the TIS11d TZF domain bound to the RNA sequence 5'-UUAUUUAUU-3' comprises a pair of novel CCCH fingers of type CX(8)CX(5)CX(3)H separated by an 18-residue linker. The two TIS11d zinc fingers bind in a symmetrical fashion to adjacent 5'-UAUU-3' subsites on the single-stranded RNA via a combination of electrostatic and hydrogen-bonding interactions, with intercalative stacking between conserved aromatic side chains and the RNA bases. Sequence specificity in RNA recognition is achieved by a network of intermolecular hydrogen bonds, mostly between TIS11d main-chain functional groups and the Watson-Crick edges of the bases. The TIS11d structure provides insights into the RNA-binding functions of this large family of CCCH zinc finger proteins.