Abstract
Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity.
Highlights
The Cys2His2 zinc finger (ZF) domain, initially discovered in TFIIIA from Xenopus laevis [1,2], is the most common DNA-binding (DB) motif in eukaryotes, and the second most frequently-encoded protein domain in the human proteome, being found in approximately 2% of all our proteins [3,4,5]
Cys2His2 ZF domains are very suitable for the construction of artificial transcription factors (TFs) as they usually bind as covalent tandem repeats, allowing the recognition of extended asymmetrical DNA sequences
The results showed no difference in binding affinity of Recombinant TFs (rTFs) with either VP16 or VP64 (Figure 2), suggesting that the activation domain (AD) in such constructs performed well, at least with respect to DNA-binding ability
Summary
The Cys2His zinc finger (ZF) domain, initially discovered in TFIIIA from Xenopus laevis [1,2], is the most common DNA-binding (DB) motif in eukaryotes, and the second most frequently-encoded protein domain in the human proteome, being found in approximately 2% of all our proteins [3,4,5]. Each ZF module holds both an anti-parallel b-sheet and a recognition a-helix that interacts directly with ,3 bps of DNA in the major groove This structurally-simple bba domain is stabilized by hydrophobic interactions and the chelation of single zinc ion between a pair of cysteines from the b-sheet and a pair of histidines from a-helix [6]. The Cys2His ZF of Zif268 has recently become known as a versatile scaffold for use in engineering novel DBDs with sequence-specificity [8,9,10] and has shown the ability to bind a variety of DNA sequences [11,12,13]. ZF proteins have been used in the fields of drug discovery [21], stem cell research [22,23], and gene targeting with sequence-specific integrases, nucleases, and methylases [17,24,25,26,27,28]
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