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.

Welcome to PPS students 2015-16!

This post is extremely like those I have written at this time of year for the past few years. This is because what I have to say now is very, very similar...

I would like to offer a warm welcome to the Principles of Protein Structure blog to all students who have just started studying Birkbeck's Principles of Protein Structure (PPS) course, and a welcome back to any who have taken a break in studies and intend to complete the course this year.

I run this blog to link the material that you will be studying in the course to new research developments in the areas of protein structure and function and related aspects of biotechnology and medicine. I might, example, report on talks given in the ISMB seminar series run jointly by the Department of Biological Sciences at Birkbeck and research departments in neighbouring University College London. The programme for Autumn 2015 focuses on transcription, which is the process through which DNA is copied into RNA; this will be covered in some detail in one of the later sections of the course, the Protein Lifecycle. Other posts may be reports from conferences or summaries of recently published papers in protein structure, protein bioinformatics and allied areas. Look out for an account of a lecture to be given at Birkbeck in October by Venki Ramakrishnan, who was awarded a share of the Nobel Prize for Chemistry in 2009 for structural studies of ribosomes and will shortly take over as President of the Royal Society.

Some posts on this blog are written by "guest blogger" Jill Faircloth, who took the MSc in Structural Molecular Biology a few years ago and is now working as a freelance science communicator. She introduces herself in this post written in March 2012, in which she also describes how she found the later part of the PPS course and her thoughts on the two choices available for the second year of the MSc.

Do, if you get a chance, look through some blog posts from earlier years to see the kind of topics that we will be discussing. However, don't be discouraged if at this stage of the course you find the science presented there difficult to understand. I can assure you that it will get easier!

I particularly recommend that you look at a couple of posts from December 2013 and July 2014 about the history of structural science, particularly X-ray crystallography. Crystallography was the first method to be developed for solving the structure of biological macromolecules, and it is still the most important. The year 2014 was designated by the United Nations as the International Year of Crystallography, marking the year between the centenaries of the publication of the first papers on X-ray diffraction and the award of the 1915 Nobel Prize for Physics to the father-and-son team of William and Lawrence Bragg who made the principal discoveries.

So - the best of luck for the 2015-16 PPS course and for your studies at Birkbeck! We hope that many of you will go on to complete our MSc in Structural Molecular Biology.

Best wishes,

Dr Clare Sansom Senior Associate Lecturer, Biological Sciences, Birkbeck and Tutor, Principles of Protein Structure

From Genome to Proteome: BCA Winter Meeting 2012

The British Crystallographic Association is the main UK organisation supporting the science of crystallography in all its forms. Every year, its Biological Structures Group holds a meeting in the run-up to Christmas to discuss and celebrate recent developments in structural biology research. In 2012, this Winter Meeting was held at the MRC Laboratory of Molecular Biology at the University of Cambridge.

The LMB, as it is usually known, is one of the birthplaces of modern structural and molecular biology. It moved into its current building in 1962, the year when four of its most famous scientists were awarded two Nobel Prizes for some of the most important discoveries in twentieth-century biology: James Watson and Francis Crick or the structure of DNA, and Max Perutz and John Kendrew for the very first three-dimensional structures of proteins (myoglobin and haemoglobin, respectively).

It was appropriate, therefore, that the theme of this year's Winter Meeting was "From Genome to Proteome". The basic molecular processes that underlie all of life - DNA replication, transcription of DNA into RNA and translation of RNA into protein - are all, now, quite well understood. These processes are all very complicated and require numerous proteins, many of which interact together to form complexes and "molecular machines" that are quite large, at least in molecular terms. Scientists presenting at the meeting discussed recent, innovative studies of the structures of many of these proteins and the nucleic acids that they interact with. Many of these processes will be discussed in some detail in section 8 of the PPS course, "The Protein Lifecycle".

The meeting programme was divided into three sections, corresponding respectively to DNA synthesis and repair, RNA transcription and protein translation.

DNA Replication and Repair

DNA synthesis and repair are not even mentioned in the famous Central Dogma of Molecular Biology (put very simplistically, DNA makes RNA makes protein) but they are, of course, essential for it. The first speaker in this session, and therefore in the meeting as a whole, was Luca Pellegrini from the University of Cambridge. He described structural studies of the first part of this process: the initiation of DNA synthesis. In all organisms, this process involves an enzyme called primase, which is found at the DNA replication fork - the point at which the strands of the original DNA helix divide so that a new strand can be synthesised on each of the template strands. Pellegrini and his group have solved the structure of several of the subunits of yeast primase, alone and bound to part of the DNA polymerase Pol alpha, and are using these structures to deduce the precise mechanism of this vitally important process.

Then Neil Kad of the University of Essex described the techniques he has developed for visualising individual molecules, and how he is applying them to the study of DNA repair by nucleotide excision. Briefly, this technique involves stretching a single molecule of DNA between two positively charged silica beads, and tagging individual molecules of DNA-binding proteins using fluorescent quantum dots so that their binding to and progress along this DNA "tightrope" can be monitored. He has discovered that although single subunits of the Uvr DNA repair protein complex may bind DNA and search it for errors, a complex between the subunits UvrA and UvrB is required for quick and efficient searching.

Schematic diagram of a "DNA tightrope" with labelled proteins bound. (c) Neil Kad, from the Kad Lab homepage

Transcription

The spliceosome is a "molecular machine" comprised of protein and small nuclear RNA (snRNA) subunits that found only in eukaryotes and that catalyses the removal of introns from the messenger RNA precursor molecules that are initially transcribed from DNA. Chris Oubridge, a member of Kiyoshi Nagai's group at the MRC Laboratory of Molecular Biology in Cambridge (and therefore one of the "home team") described an atomic resolution structure of a complex known as U1 that forms a major part of the soliceosome. This "small nuclear ribonucleoprotein" (snRNP) comprises the snRNA molecule U1 bound to ten proteins. This technically challenging exercise in X-ray crystallography is yielding important insights into the function and mechanism of this important part of the spliceosome.

Structure of the U1 ribonucleoprotein, from Kiyoshi Nagai's web pages at the MRC-LMB.

Another interesting presentation in the Translation section was given by David Lilley from the University of Dundee, who described the structures of kink turns in RNA molecules, and how these structural motifs interact with proteins.

Translation

Since the modern Laboratory o Molecular Biology was constituted as the "Unit for Research on the Molecular Structure of Biological Systems'" in 1947, nine Nobel prizes have been awarded to scientists working there. Its most recent laureate, Venki Ramakrishnan, shared the 2009 chemistry prize with Tom Steitz from the US and Ada Yonath from Israel for determining the first atomic resolution structure of the ribosome. Israel Sanchez from Ramakrishnan's lab at the LMB gave a presentation on the mechanism by which stop codons, which give the signal to terminate protein synthesis, are decoded on the ribosome. This process, which occurs when one of the stop codons (UAA, UAG and UGA in the standard genetic code) binds to the ribosomal A site, is still less well understood than the process through which "sense" codons are decoded into amino acids. Sanchez and his colleagues are studying the structure and function of ribosomes bound to modified RNA in which the uridine in the first position of a stop codon has been substituted by pseudo-uridine. They have discovered that the decoding centre of the ribosome is more flexible than they had originally thought, an insight that may help the understanding of the termination of protein synthesis further.

The final speaker was Birkbeck's own Cara Vaughan. She discussed some of her recent research using a combination of X-ray crystallography and electron microscopy to decipher the assembly of the kinetochore. This is a structure that forms in eukaryotic cells during cell division and that links the dividing chromosome to the mitotic spindle. Vaughan's research concerns a protein called Hsp90 that activates many signalling proteins. This protein is a member of a class of proteins termed the chaperones, which are generically involved in the folding, unfolding and activation of other proteins. Vaughan and her co-workers have solved the structure of two interacting proteins found in yeast, Sgt1 and Skp1, which togethe3r seem to hold Hsp90 in an open conformation that enables other kinetochore proteins to bind.

Image of a dividing eukaryotic cell. The chromosomes are shown in blue, the microtubules of the mitotic spindle in green, and the kinetochores in pink. Image from Wikimedia Commons.

The annual Winter Meeting is the most high profile event organised by the Biological Structures Group of BCA. The association as a whole organises many other events, including, this year, the annual European Crystallographic Meeting. ECM 28 will be held at the University of Warwick from 25-29 August 2013; it will provide an opportunity for British and European crystallographers to celebrate the origin of their science with the discovery of X-ray diffraction by father and son William Henry and William Lawrence Bragg, almost exactly a hundred years ago.