A transcription factor that needed zinc
Aaron Klug’s laboratory, working at the Medical Research Council Laboratory of Molecular Biology in Cambridge, was trying to understand a protein called transcription factor IIIA, which the African clawed frog Xenopus laevis uses to help make the RNA component of its ribosomes. The protein turned out to need zinc ions to do its job, an unusual dependency for a protein that binds DNA, and no other regulatory protein had been reported to need zinc in this way before. When Klug and colleagues examined the amino acid sequence of TFIIIA, they found the same short block of amino acids repeated nine times along the protein, each repeat carrying the same fixed pair of cysteine and histidine residues. Modelling that repeat led them to propose a specific structure: a small loop of protein folded around a single zinc ion, which they named the zinc finger.
A repeated pattern in the sequence
The zinc requirement was established by testing the protein’s activity with and without available zinc, and by using spectroscopic methods, including extended X-ray absorption fine structure, to identify exactly which atoms of the protein were coordinating each zinc ion, confirming a pairing of two cysteines and two histidines around each metal. Klug’s group then used the repeated sequence pattern to build a structural model of how each repeat might fold, proposing that the zinc-stabilised loop presented a short helix positioned to make direct, sequence-specific contact with the DNA double helix. It took several more years, and further crystal structures of zinc finger proteins bound to their DNA targets published in the early 1990s, for that modelled arrangement to be confirmed atom by atom.
Modelling the finger
The zinc finger model held up in full: crystal structures showed exactly the arrangement Klug’s group had proposed, with a short helix from each finger sitting in the major groove of the DNA and its side chains reading out particular bases, while successive fingers along the protein’s length line up to cover a longer stretch of sequence. The motif turned out to be far from a one-off feature of TFIIIA; related cysteine-and-histidine zinc fingers are now known to occur across a large share of the proteins encoded in the human genome, doing everything from binding DNA and RNA to helping proteins fold and interact with one another. Its modular, repeatable structure is now a standard feature described throughout structural biology.
Confirming how it grips DNA
What the original work did not do is explain why this particular solution, a small zinc-stabilised loop rather than some other structural strategy, is used so widely across such different proteins and organisms, a question that structural and evolutionary biology have continued to work on since. Nor did the discovery of the motif immediately reveal every practical use it might have; the idea of stringing engineered zinc fingers together to target a chosen DNA sequence, and fusing them to a cutting enzyme to edit a genome at that exact spot, developed over the following decade as a separate line of protein engineering, not as part of Klug’s original structural finding.
From a natural motif to an editing tool
Because each zinc finger can be swapped for another with a different DNA-reading preference, researchers realised the motif could be treated like a set of interchangeable parts: arrays of several fingers, strung together, can be designed to recognise a chosen stretch of DNA that is unlikely to occur anywhere else in a large genome. Fusing such an array to a DNA-cutting enzyme produced zinc finger nucleases, an early genome-editing tool used to make targeted changes in the DNA of organisms ranging from plants to human cells, including experimental attempts to disrupt a gene involved in HIV infection. The same fingers, fused instead to a gene-activating or gene-silencing protein, are also used to switch particular genes on or off.
Why a small structural motif is worth knowing
This is a useful one for understanding how structural biology feeds engineering: a specific, almost incidental observation about a frog’s ribosome-making machinery turned out to describe a general-purpose molecular shape that biology reuses constantly and that biotechnology has since learned to redesign on demand. It rewards attention less for drama than for the clean logic of the chain, an odd zinc requirement, a repeated sequence, a proposed fold, a confirmed structure, and eventually a toolkit, each step building directly on the last. An hour with it is time well spent for anyone who wants to see how a basic-research puzzle in the 1980s underwrites gene-editing tools still used today.