Thursday, 19 November 2015

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.