Bernal Lecture 2015: Terminating Protein Synthesis

Professor J.D. Bernal, known to his colleagues and contemporaries as ‘Sage’, spent much of his career as a professor of Physics at Birkbeck and became the first chair of the Department of Crystallography in 1963. When he retired in 1968 the college founded a series of lectures in his honour. The first of the Bernal Lectures, in 1969, was given by Dorothy Hodgkin, winner of the 1964 Nobel Prize for Chemistry for solving the structures of ‘important biological substances’, mainly penicillin and vitamin B12. In 2015 we were honoured to welcome another Chemistry Nobel Laureate to give this annual lecture. Professor Sir Venki Ramakrishnan of the MRC Laboratory of Molecular Biology in Cambridge was awarded the prize in 2009 with Thomas Steitz of Yale University, USA and Ada Yonath of the Weizmann Institute of Science, Israel, for studies of the structure and function of the ribosome: the ‘molecular machine’ that catalyses the synthesis of proteins from their messenger RNA (mRNA) templates.

The lecture, held on 19 October 2015, was introduced by David Latchman, Master of Birkbeck College and Professor of Genetics. He welcomed three generations of Bernal’s descendants to Birkbeck, highlighted the success of the lecture series in attracting some of the most distinguished researchers in structural biology and allied disciplines, and explained that the topic of the lecture overlapped with some of his own research interests in the regulation of gene expression.

Prof. Ramakrishnan began his lecture by explaining that protein synthesis was a complex process, involving many proteins as well as the ribosome itself, and that he would be talking about a particular point in this process: namely, how it ends (formally, the termination of protein translation). He showed an image of a ribosome in the process of protein synthesis that, he explained, represented the culmination of 40 years’ work on its structure, and explained how the linear mRNA molecule wound through a cleft between the two subunits of the ribosome. As the mRNA passes through the ribosome each of its three-base ‘codons’ comes into contact with three sub-parts of the ribosome’s active site – the A-site, P-site and E-site – in turn. When a codon enters the A-site it binds to the anti-codon of the transfer RNA (tRNA) carrying the next amino acid; the amino acid is bonded to the previous amino acid in the growing protein chain in the P-site, and the now empty tRNA released from the ribosome in the E-site. This continues until the new protein chain is complete. This is signalled by one of the so-called ‘stop codons’ UGA, UAG and UAA, which have no corresponding tRNAs, entering the ribosome’s A-site. The new protein is released from the ribosome to fold into its native structure, and the ribosome subunits dissociate.

Diagram of a ribosome showing the three tRNA binding sites during protein elongation
Taken from the PDB ‘Structural View of Biology: The Ribosome’
© David Goodsell, 2010

Although the process of adding amino acids to a protein chain is extremely similar in all organisms, there are significant differences between bacteria (prokaryotes) and eukaryotes in the process of termination – as, indeed, there are in the initiation of protein synthesis. We are beginning to understand these mechanisms only now that we can obtain high resolution structures of ‘snapshots’ of the ribosome at different points during the protein synthesis cycle and follow the sequence of conformational changes that occur then.

All stop codons are recognised and decoded by proteins known as a release factors. Bacteria have two of these: RF1 recognises UAG, RF2 recognises UGA and they both recognise UAA. Eukaryotes have only one RF, which can recognise all these codons. These three proteins all have a common sequence motif, GGQ, which is known to be involved in the release of the protein from the ribosome. The structures of the eukaryotic and prokaryotic release factors are different, but all bind to the ribosome in such a way that the GGQ motif and the part of the structure that recognises the stop codon are exactly the same distance apart as the length of a tRNA molecule. These parts of the protein will therefore interact with the peptide and the stop codon at the same time.

Prof. Ramakrishnan and his group spent years trying to obtain near atomic resolution structures of functional ribosome-release factor complexes; this problem was solved initially for the smaller prokaryotic ribosomes but now for eukaryotic ones as well. In all cases, the release factors bind to the ribosome in a different way to the tRNA molecules, inducing a different conformational change in the complex. Using the bacterial structures, the group was able to understand why the factors RF1 and RF2 only recognise the codons that they do.

In the case of eukaryotes, not only were the structures harder to obtain, but the basic question to be asked was more complex: how can a single protein recognise the stop codons UGA, UAG and UAA, but not UGG (which codes for the amino acid tryptophan)? Ramakrishnan reasoned that mutating the GGQ motif in the release factor would make it inactive, and that binding this mutant protein to the ribosome with an ATPase might ‘trap’ the complex in the structure that it takes up before protein is released and allow the structure to be determined. Electron micrographs of these structures have shown that the three anti-codons and no others are recognised through a combination of base stacking and hydrogen bonding. Ramakrishnan ended his talk by comparing anti-codon binding to a NAND gate in electronics, with G representing ‘1’ and A ‘0’: any combination except GG (‘11’) in the second and third anti-codon positions leads to termination of translation and protein release.

This work was published in Nature in August 2015 and the (very large!) structures of the complexes – one snapshot with the release factor bound in each of the ribosome subsites – are available in the PDB as entries 3JAG,3JAH and 3JAI. These are some of the most recent of the 103 PDB structures on which Prof Ramakrishnan has so far been named as an author; you can view them all on a timeline on the PDB site.

Note: If you are reading this blog post as a current PPS student, don’t be surprised if you find it difficult to understand. We will cover the structure and mechanism of the ribosome later in the course (in Section 8: The Protein Lifecycle). If you bookmark this blog post and come back to it after you have studied that section you should find that you can make much more out of it.

Science Week 2014: Birkbeck and the History of Crystallography

Science Week at Birkbeck in 2014 featured two lectures on Department of Biological Sciences, both presented on 2 July. One of these was a double act from two distinguished emeritus professors and Fellows of the College, Paul Barnes and David Moss. Remarkably, they both started their working lives at Birkbeck on the same day – 1 October 1968 – and so had clocked up over 90 years of service to the college between them by Science Week 2014.

The topic they took was a timely one: the history of the science of crystallography over the past 100 years. UNESCO has declared 2014 to be the International Year of Crystallography in recognition of the seminal discoveries that started the discipline, which were made almost exactly 100 years ago; a number of the most important discoveries of that century were made by scientists with links to Birkbeck.

The presenters divided the “century of crystallography” into two, with Barnes speaking first and covering the first 50 years. In giving his talk the title “A History of Modern Crystallography”, however, he recognised that crystals have been observed, admired and studied for many centuries. What changed at the beginning of the last century was the discovery of X-ray diffraction. Wilhelm Röntgen was awarded the first-ever Nobel Prize for Physics for his discovery of X-rays in 1896, but it was almost two decades before anyone thought of directing them at crystals. The breakthroughs came when Max von Laue showed that a beam of X-rays can be diffracted by a crystal to yield a pattern of spots, and the father-and-son team of William Henry and William Lawrence Bragg showed that it was possible to derive information about the atomic structure of crystals from their diffraction patterns. These discoveries also solved – to some extent – the debate about whether X-rays were particles or waves, as only waves diffract; we now know that all electromagnetic radiation, including X-rays, can be thought of as both particles and waves

Von Laue and the Braggs were awarded Nobel Prizes for Physics in 1914 and 1915 respectively, and between 1916 and 1964 no fewer than 13 more Nobel Prizes were awarded to 18 more scientists for discoveries related to crystallography. Petrus Debye, who won the Chemistry prize in 1936, showed how to quantify the thermal motion of atoms as vibrations within a crystal. He also invented one of the first powder diffraction cameras, used to obtain diffraction patterns from powders of tiny crystallites. Another Nobel Laureate, Percy Bridgman, studied the structures of materials under pressure: it has been said that he would “squeeze anything he could lay his hands on”, often up to intense pressures.

Scientists and scientific commentators often argue about which of their colleagues would have most deserved to win the ultimate accolade. Barnes named three who, he said, could easily have been Nobel Laureates in the field of crystallography. One, Paul Ewald, was a theoretical physicist who had studied for his PhD under von Laue in Munich, and the other two had strong links with Birkbeck. JD “Sage” Bernal was Professor of Physics and then of Crystallography here; he was famous for obtaining, with Dorothy Crowfoot (later Hodgkin) the first diffraction pattern from a protein crystal, but his insights into the atomic basis of the very different properties of carbon as diamond and as graphite were perhaps even more remarkable. He took on Rosalind Franklin, whose diffraction patterns of DNA had led Watson and Crick to deduce its double helical structure, after she left King’s College, and she did pioneering work on virus structure here until her premature death in 1958.

Barnes ended his talk and led into Moss’s second half-century with a discussion of similarities between the earliest crystallography and today, as now, you only need three things to obtain a diffraction pattern: a source of X-rays, a crystalline sample, and a recording device; the differences all lie in the power and precision of the equipment used. He demonstrated this with a “symbolic demo” that ended when he pulled a model structure of a zeolite out of a large cardboard box.

Paul Barnes demonstrates the basic principles of X-ray crystallography using a large cardboard box. Photo © Harish Patel and Ruben Zamora, Department of Psychological Sciences, Birkbeck

David Moss then took over to describe some of the most important crystallographic discoveries from the last half-century. His talk concentrated on the structures of large biological molecules, particularly proteins, and he began by explaining the importance of protein structure. All the chemistry that is necessary for life is controlled by proteins, and knowing the structure of proteins enables us to understand, and potentially also to modify, how they work.

Even the smallest proteins contain thousands of atoms; in order to determine the position of all the atoms in a protein using crystallography you need to make an enormous number of measurements of the positions and intensities of X-ray spots. The process of solving the structure of a protein is no different from that of solving a small molecule crystal structure, but it is more complex and takes much more time. Very briefly, it involves crystallising the protein; shining an intense beam of X-rays on the resulting crystals to produce diffraction patterns, and then doing some extremely complex calculations. The first protein structures, obtained without the benefit of automation and modern computers, took many years and sometimes even decades.

Thanks to Bernal’s genius, energy and pioneering spirit, Birkbeck was one of the first institutes in the UK to have all the equipment that was needed for crystallography. This included some of the country’s first “large” computers. One of the first electronic stored-program computers was developed in Donald Booth’s laboratory here in the 1950s. In the mid-1960s the college had an ATLAS computer with a total memory of 96 kB. It occupied the basements of two houses in Gordon Square, and crystallographers used it to calculate electron density maps of small molecules. Protein crystallography only “took off” in the 1970s with further improvements in computing and automation of much of the experimental technique.

Today, protein crystallography can almost be said to be routine. The first step, crystallising the protein, can still be an important bottleneck, but data collection at powerful synchrotron X-ray sources is extremely rapid and structures can be solved quite easily with user-friendly software that runs on ordinary laptops. There are now over 100,000 protein structures freely available in the Protein Data Bank (PDB), and about 90% of these were obtained using X-ray crystallography. The techniques used to obtain the other 10,000 or so, nuclear magnetic resonance and electron microscopy, are more specialised.

Moss ended his talk by describing one of the proteins solved in his group during his long career at Birkbeck: a bacterial toxin that is responsible for the disease gas gangrene (PDB 1CA1). This destroys muscle cells by punching holes in their membranes, and its victims usually have to have limbs amputated to save their lives. Knowing the structure has allowed scientists to understand how this toxin works, which is the first step towards developing drugs to stop it. But you can learn even more about how proteins work if you also understand how they move. Observing and modelling protein motion in “real time” still poses many challenges for scientists as the second century of crystallography begins.

Structure of alpha-toxin, the key Clostridium perfringen toxin in gas gangrene. Image from the PDB.

A Very Short History of Crystallography

You might possibly have been intrigued to read in my last post that 2014 has been designated as the International Year of Crystallography. This year was chosen to celebrate the fact that this discipline - the study of atomic and molecular structure through their crystal forms - is now almost exactly a hundred years old. Admittedly, the first paper in the discipline, rather charmingly titled just "The diffraction of short electro-magnetic waves by a crystal" was published in 1913, and the Nobel Prize awarded two years later, but 2014 is at least a good compromise.

It can be said, perhaps simplistically, that crystallography was invented by the father-and-son team of William Henry and William Lawrence (known as Lawrence) Bragg, at the Universities of Leeds and Cambridge in the UK. The Braggs, however, did not aim to found a new discipline or even to investigate the atomic properties of matter. They were more interested in solving a problem that had been puzzling the cleverest physicists in the world for almost two decades. X-rays had been discovered by Wilhelm Rőntgen in Germany in 1895, but their very name (the unknown X) suggests reveals their controversial nature. Were they particles or waves?

The older Bragg, William, was convinced that X-rays were particles, and set out to prove this to his son (who favoured the wave theory) by exploiting the discovery of another German physicist, von Laue, that X-rays shone at a crystal were scattered and could produce a pattern on a film. Lawrence was the first to realise that these patterns could be explained by the theory that the X-rays were reflected from planes of atoms in the crystal and interfered with each other.

Lawrence presented these results to the Cambridge Philosophical Society late in 1912 and published them in the paper mentioned above the following year. This paper also included the first formulation of one of the best known of all laws of physics: Bragg's Law. This relates the wavelength of incoming X-rays and the angles that they are scattered (diffracted) to the spacing between planes of atoms in a crystal, enabling scientists to determine the geometry of atomic crystal lattices.

The Braggs worked together in Leeds and published their first structures, including that of sodium chloride (common salt) before Lawrence was sent to France to fight in the First World War. He was in the trenches when he heard that he and his father had been awarded the 1915 Nobel Prize for Physics; that news reached him shortly after that of the death of his brother Robert. At only 25, he was (and still remains) the youngest ever recipient of a Nobel Prize.

Technical advances between the wars enabled scientists working in this new discipline to solve the structures of rather more complex molecules. Kathleen Lansdale, one many women who began their research careers as the Braggs' students, solved the structures of benzene derivatives and was the first to see that aromatic rings were flat. And two later developments paved the way for the explosion in structural science that characterised the later twentieth century. In 1934, John Desmond (J.D.) Bernal, who later became the first head of the School of Crystallography at Birkbeck (the predecessor department of our Biological Sciences) and his student Dorothy Crowfoot (later Hodgkin) obtained the first X-ray diffraction patterns from protein crystals. And in the following year Lindo Patterson developed a function that greatly simplified the mathematics involved in structure determination.

Even fifty years ago however, solving crystal structures was a long and at times tedious business. A typical crystallogaphy PhD thesis of the 1960s or 1970s would contain the structures of maybe three small or medium-sized molecules. It is now possible to generate as many in a few hours, so it is possible to see clearly how structures of molecules respond to changes in conditios such as temperature and pressure.

All these discoveries have been made possible by advances in technology, and particularly by the development of synchrotron radiation as a source of powerful beams of X-rays. Synchrotron radiation is produced when charged particles are accelerated radially, and synchrotrons built primarily as X-ray sources were first built in the 1980s. The UK's synchrotron, Diamond at Harwell in Oxfordshire, is currently the fifth largest in the world. It has 23 separate "beamlines", each providing a beam of X-rays with properties that have been optimised for a particular experimental technique.

Synchrotrons provide facilities for solving structures from single crystals of large and small molecules, including, of course, proteins, and from micro-crystalline samples (the latter technique is known as powder diffraction). Although structural biology attracts much of the attention (see almost all the other posts on this blog) structures of smaller molecules can still provide important insights. Sandy Blake, a crystallographer at the University of Nottingham, is using Diamond beamlines to solve the structures of novel materials called metal-organic frameworks or MOFs that are able to store gases including hydrogen (which is a potential fuel source) and greenhouse gases.

At 100, crystallography is still a young discipline but it has radically transformed many other areas of science and, through them, the world we inhabit today. This has been reflected in decisions made by the Nobel committees over the decades. The International Union of Crystallography maintains a list of Nobels awarded for ‘achievements directly related to, or involving the use of, crystallography’. There are now 29 of these, and the latest year with no crystallography-related Nobel was 2008. Even the 2013 Chemistry prize, awarded to Martin Karplus, Michael Levitt and Arieh Warshel, appears on the list: their discipline of computational chemistry would be impossible without structural knowledge obtained through crystallography.

And it almost goes without saying that protein structure, and structural biology more generally - the disciplines taught in this course and its associated MSc - owe their existence to the development of X-ray crystallography.

This blog post is based on an article I wrote for the Royal Society of Chemistry's membership journal, Chemistry World. It will be published in the January 2014 issue of the journal.