The maize ID1 flowering time regulator is a zinc finger protein with novel DNA binding properties

The INDETERMINATE protein, ID1, plays a key role in regulating the transition to flowering in maize. ID1 is the founding member of a plant-specific zinc finger protein family that is defined by a highly conserved amino sequence called the ID domain. The ID domain includes a cluster of three different types of zinc fingers separated from a fourth C2H2 finger by a long spacer; ID1 is distinct from other ID domain proteins by having a much longer spacer. In vitro DNA selection and amplification binding assays and DNA binding experiments showed that ID1 binds selectively to an 11 bp consensus motif via the ID domain. Unexpectedly, site-directed mutagenesis of the ID1 protein showed that zinc fingers located at each end of the ID domain are not required for binding to the consensus motif despite the fact that one of these zinc fingers is a canonical C2H2 DNA binding domain. In addition, an ID1 in vitro deletion mutant that lacks the extra spacer between zinc fingers binds the same 11 bp motif as normal ID1, suggesting that all ID domain-containing proteins recognize the same DNA target sequence. Our results demonstrate that maize ID1 and ID domain proteins have novel zinc finger configurations with unique DNA binding properties.     

Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins

In previous studies, we have developed a technology for the rapid construction of novel DNA-binding proteins with the potential to recognize any unique site in a given genome. This technology relies on the modular assembly of modified zinc finger DNA-binding domains, each of which recognizes a three bp subsite of DNA. A complete set of 64 domains would provide comprehensive recognition of any desired DNA sequence, and new proteins could be assembled by any laboratory in a matter of hours. However, a critical parameter for this approach is the extent to which each domain functions as an independent, modular unit, without influence or dependence on its neighboring domains. We therefore examined the detailed binding behavior of several modularly assembled polydactyl zinc finger proteins. We first demonstrated that 80 modularly assembled 3-finger proteins can recognize their DNA target with very high specificity using a multitarget ELISA-based specificity assay. A more detailed analysis of DNA binding specificity for eight 3-finger proteins and two 6-finger proteins was performed using a target site selection assay. Results showed that the specificity of these proteins was as good or better than that of zinc finger proteins constructed using methods that allow for interdependency. In some cases, near perfect specificity was achieved. Complications due to target site overlap were found to be restricted to only one particular amino acid interaction (involving an aspartate in position 2 of the alpha-helix) that occurs in a minority of cases. As this is the first report of target site selection for designed, well characterized 6-finger proteins, unique insights are discussed concerning the relationship of protein length and specificity. These results have important implications for the design of proteins that can recognize extended DNA sequences, as well as provide insights into the general rules of recognition for naturally occurring zinc finger proteins.     

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。     

Sequence-enabled reassembly of beta-lactamase (SEER-LAC): a sensitive method for the detection of double-stranded DNA

This work describes the development of a new methodology for the detection of specific double-stranded DNA sequences. We previously showed that two inactive fragments of green fluorescent protein, each coupled to engineered zinc finger DNA-binding proteins, were able to reassemble an active reporter complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), was demonstrated in vitro to produce a DNA-concentration-dependent signal. Here we endow the SEER system with catalytic capability using the reporter enzyme TEM-1 beta-lacatamase. This system could distinguish target DNA from nontarget DNA in less than 5 min, representing a more than 1000-fold improvement over our previous SEER design. A single base-pair substitution in the DNA binding sequence reduced the signal to nearly background levels. Substitution of a different custom zinc finger DNA-binding domain produced a signal only on the new cognate target. Signal intensity was not affected by genomic DNA when present in equal mass to the target DNA. These results present SEER as a rapid and sensitive method for the detection of double-stranded DNA sequences.     

Role of Conserved Residues of the WRKY Domain in the DNA-binding of Tobacco WRKY Family Proteins

Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn²⁺. Substitution of the conserved cysteine and histidine residues of the plant-specific C₂H₂-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.     

Zinc fingers and other domains cooperate in binding of Drosophila sry beta and delta proteins at specific chromosomal sites.

The closely related Drosophila serendipity (sry) beta and delta zinc finger proteins display consensus in vitro DNA recognition sequences differing by 4 of 13 nucleotide positions and bind in vivo to distinct sets of sites on polytene chromosomes. We compared the pattern of in vivo chromosomal binding of deleted forms of the sry delta protein fused to beta-galactosidase and expressed in Drosophila transgenic lines. Results show that the carboxy-terminal DNA-binding finger domain is required and sufficient for binding at specific chromosomal sites but that this binding does not nearly reproduce the wild-type pattern. An NH2-terminal domain of the sry delta protein is essential to its specificity of in vivo interaction with chromatin. In vitro and in vivo experiments using reciprocal finger swap between the sry beta and delta proteins suggest that the in vivo specificity is dependent on selective protein-protein contacts at defined chromosomal sites, in addition to DNA specific recognition.     

CCHX zinc finger derivatives retain the ability to bind Zn(II) and mediate protein-DNA interactions

Classical (CCHH) zinc fingers are among the most common protein domains found in eukaryotes. They function as molecular recognition elements that mediate specific contact with DNA, RNA, or other proteins and are composed of a betabetaalpha fold surrounding a single zinc ion that is ligated by two cysteine and two histidine residues. In a number of variant zinc fingers, the final histidine is not conserved, and in other unrelated zinc binding domains, residues such as aspartate can function as zinc ligands. To test whether the final histidine is required for normal folding and the DNA-binding function of classical zinc fingers, we focused on finger 3 of basic Krüppel-like factor. The structure of this domain was determined using NMR spectroscopy and found to constitute a typical classical zinc finger. We generated a panel of substitution mutants at the final histidine in this finger and found that several of the mutants retained some ability to fold in the presence of zinc. Consistent with this result, we showed that mutation of the final histidine had only a modest effect on DNA binding in the context of the full three-finger DNA-binding domain of basic Krüppel-like factor. Further, the zinc binding ability of one of the point mutants was tested and found to be indistinguishable from the wild-type domain. These results suggest that the final zinc chelating histidine is not an essential feature of classical zinc fingers and have implications for zinc finger evolution, regulation, and the design of experiments testing the functional roles of these domains.