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Refining the structure of zinc fingers


Phage display and solving the 'mystery' of the stereochemical code
Aaron Klug Scientist
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So Yen Choo started using... perio-phage display to try to build up... a repertoire of sequences of zinc fingers which would bind given sequences of DNA and the way we did that was to fix... to take the Z1F268 which we based it on and change the middle DNA triplet into the desired binding site. And so, we had two fingers, fingers one and three, binding to the DNA sites one and three and the DNA target, that was put into the DNA sequencing... the DNA binding site, subsite that should be, into subsite and then we varied the DNA sequence just because we didn't know how representative the crystal structure was, in all nine positions of the recognition helix. And we found miraculously that all the selected binding sites all basically we got consensus of binding in the positions minus one, three and six which was rather very gratifying. But you see you have to know a structure. Now these libraries weren't so big as we were varying... we were varying nine positions and our libraries they only had, at that time, 107 or 108 members. But later on, we built bigger libraries and varied a smaller number of sites, only four sites, and not just positions minus one, three and six, but minus one, two and six. Minus one, two, three and six and I'll come to say how site two was involved.

But in the meantime, in the laboratory, Louise Ferrel who was the assistant of Daniela Rhodes and they started working on zinc fingers and Louise tried to crystallise a zinc finger peptide. There were only two-zinc fingers, it's from Tram Track which is a gene in drosophila which was being worked on by Andrew Travis in one of the other divisions. And Louise managed to crystallise this, binding to DNA and the structure was solved and published in Nature in 1993. Which was about 18 months after the pair position paper. Now what this showed were two things. The one was it broadened the amino acids which would bind cells: for example, it showed that you could... that if you had an adenine which wasn't represented in the Z1F268 structure, then you had to have an asparagine to bind to it and so it extended the code. But the other thing it showed very clearly, something we had suspected from our phage display, but we found with our phage display experiments, there were certain positions which we couldn't determine because it was clear they were being coupled to something else. And so, the structure not only gave us some new elements in the stereochemical code but solved the mystery. What the structure showed very clearly was that, from position two in the recognition helix there was an interaction with a site on the second strand of the DNA which was part of the... sorry base of the second strand of the DNA which was part of the subsite of the preceding finger. So in other words there was a... it was an interesting business because that site, that DNA position had to... was part of a double helical and so its partner had to be recognised by the preceding finger. So what this meant, a very important discovery, it meant that although the fingers were structurally independent they were synergistically involved and that made a big difference. But there was still nevertheless... it was still one amino acid per one... one amino acid per one DNA... sorry to one base. Now I didn't say this before... but in the many transcription factors being worked which all... where there's homodimers or variant heterodimers it isn't like that. The interface between the DNA recognition helix and the DNA is more complex. In most cases there's one amino acid interact with more than one base and conversely one base can interact with more than... more than one amino acid, so we haven't got this simple stereochemical code, we've got zinc fingers, even with this extra interaction to the second strand it's still one amino acid per... per base and vice versa and one base per amino acid. Now this meant, and it was at this point, that I decided that really this is a marvellous system. We should be able to develop it for intervening in gene regulation. Particularly as once we'd worked out the stereochemical code, so it was a very important discovery, the second crystal structure.

Born in Lithuania, Aaron Klug (1926-2018) was a British chemist and biophysicist. He was awarded the Nobel Prize in Chemistry in 1982 for developments in electron microscopy and his work on complexes of nucleic acids and proteins. He studied crystallography at the University of Cape Town before moving to England, completing his doctorate in 1953 at Trinity College, Cambridge. In 1981, he was awarded the Louisa Gross Horwitz Prize from Columbia University. His long and influential career led to a knighthood in 1988. He was also elected President of the Royal Society, and served there from 1995-2000.

Listeners: Ken Holmes John Finch

Kenneth Holmes was born in London in 1934 and attended schools in Chiswick. He obtained his BA at St Johns College, Cambridge. He obtained his PhD at Birkbeck College, London working on the structure of tobacco mosaic virus with Rosalind Franklin and Aaron Klug. After a post-doc at Childrens' Hospital, Boston, where he started to work on muscle structure, he joined to the newly opened Laboratory of Molecular Biology in Cambridge where he stayed for six years. He worked with Aaron Klug on virus structure and with Hugh Huxley on muscle. He then moved to Heidelberg to open the Department of Biophysics at the Max Planck Institute for Medical Research where he remained as director until his retirement. During this time he completed the structure of tobacco mosaic virus and solved the structures of a number of protein molecules including the structure of the muscle protein actin and the actin filament. Recently he has worked on the molecular mechanism of muscle contraction. He also initiated the use of synchrotron radiation as a source for X-ray diffraction and founded the EMBL outstation at DESY Hamburg. He was elected to the Royal Society in 1981 and is a member of a number of scientific academies.

John Finch is a retired member of staff of the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK. He began research as a PhD student of Rosalind Franklin's at Birkbeck College, London in 1955 studying the structure of small viruses by x-ray diffraction. He came to Cambridge as part of Aaron Klug's team in 1962 and has continued with the structural study of viruses and other nucleoproteins such as chromatin, using both x-rays and electron microscopy.

Tags: Louise Ferrel, Yen Choo, Daniela Rhodes, Andrew Travis

Duration: 5 minutes, 58 seconds

Date story recorded: July 2005

Date story went live: 24 January 2008