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More work and papers on TF3A


Work on TF3A with Jonathan Miller
Aaron Klug Scientist
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I had a new research student coming, a chap called Jonathan Miller who had a degree from Yale and also a higher diploma in biophysics from them, he turned out to be a very good mathematician, he landed up... it's an interesting story himself, he used to walk around with books on abstract mathematics, I don't know if you remember him, John? He could never decide what he wanted to do and just as an aside I discovered later on he'd been enrolled as a PhD student here in Cambridge and I was his supervisor, but in his third year he was also enrolled in Princeton physics without telling me... because he still then couldn't decide, I think he's rather sorry now because he could get onto a good thing if he had realised where this was going to lead. So he had had practical experience although he was good at mathematics, both his parents were doctors and they wanted him to work in medicine or biomedicine so he'd spent every summer in the last four years working in labs so he knew how to do bio chemical operations but he was very terribly messy, very messy, one of the messiest people I've ever known... never cleared up, which figures in the story later. So I said, well, the thing to do, you can follow the recipe and there should be... and so we began to make TF3A, there were recipes for this and it was interesting, none of the people later on, Louise Fairall and Daniella joined... and they couldn't bear themselves to kill a frog so I had to pith the frogs and kill them because I used to do that when I was a medical student and extract the oocytes. So they followed the recipe and they got very low yields, looking at gels where you could run the protein on, it was all described by Reuter and Brown and the binding sites. We got very low yields so there was clearly something wrong with it, so I thought obviously the extraction procedure they describe are very inefficient, but you see those two were molecular biologists and they work with gels and they work in microgram quantities, we wanted to work in milligram quantities, which we knew to be there. Because Hugh Pelham had shown me gels which show that there must be able to be milligrams... you should be able to get dozens of milligrams from a set of ovaries which were ovulated. So now we were going to change the buffer conditions and now I looked at their buffers and what they had done was to use EDTA which is a chelating agent which takes up metals, which was very sensible to do because if you have any metals around, you're working with RNA or DNA they tend to hydrolyse the nucleic acid. So they put in EDTA.

Now the sequence of the protein wasn't known, the sequence of the gene wasn't known, TF3A, but amino acid analysis was known, was known to contain quite a lot of cysteines and histidines. Now cysteines, there were a lot of... there were... about 20 cycsteines in it so they assumed there must be SS bridges in it, so they put in reducing agents, dithiothreitol to protect it and so the... now both of these things were removing metals, because dithiothreitol because of the sulphur groups in it will also, and in fact has a binding constant for metals, for zinc which turned out to be the metal, was ten to the ten, ten to the eleven for other metals as well. So what they were doing, and we showed that what was happening was the... we were trying to make a complex of the TF3A and the DNA target, we knew the sequence of the DNA target, everything was known, it was well set up problem, the things associated, so we gradually changed the buffer conditions and I came to the conclusion that there couldn't be any SS bridges because it didn't make any difference. And indeed, sorry I won't describe everything, let me go back a bit. I thought there'd be a test if there's SS bridges, so I knew enough chemistry to know that sodium borohydride was a very strong reducing agent so I got Jonathan to add sodium borohydride to the TF3A, it had no effect at all and this meant that there couldn't be any SS bridges in it. So the only thing it could be would be a metal. And so what we did was to gradually omit the dithiothreitol, start omitting EDTA, we began to get higher yields. That was just... but by this time I deduced there must be a metal there. So then to find out what the metal was we did send the material to a lab in medical school who were involved in, had absorption spectral photometry to test metals in different specimens, but the sub wasn't pure enough, we had to purify it still further... so what we did eventually to... it's quite a long story, was to start adding EDTA specifically, titrating it and using other collating agents for example phenthramine which takes out copper and other things which take out iron to see if they acted. But the EDTA and these things, we found that... and then added metals back to the preparation that Jonathan was doing, titrate metals back and we found that zinc and only zinc... when added back was able to bind back to... in other words, it restored binding to DNA which was the target and also to RNA.

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: Daniela Rhodes, Jonathan Miller, Louise Fairall, Hugh Pelham

Duration: 6 minutes, 55 seconds

Date story recorded: July 2005

Date story went live: 24 January 2008