Cryo-EM analysis of ribosomal frameshifting
Lead Research Organisation:
University of Oxford
Department Name: Wellcome Trust Centre for Human Genetics
Abstract
Cell proteins are encoded in DNA, the cell's genetic material, and expressed from so-called messenger RNA copied from DNA. This process of expression involves reading a triplet nucleotide code and translating it into an amino acid polymer, the protein. The process of translation involves structures called ribosomes. These move along the messenger RNA decoding the triplets and adding one amino acid for each triplet to the growing amino acid chain. Viruses that infect cells carry their own genetic material and occasionally, they make messenger RNAs that contain a specific signal (called a frameshift signal) that tells the ribosome to stop making one type of triplet and to start making another. The result is that one messenger RNA can make two proteins. This is typical of viruses; as their own genetic material is usually relatively small, they use all sorts of tricks to maximise the number of proteins they can make. In this project, we wish to examine what happens to the ribosome when it encounters a frameshift signal. The question is 'what happens to the ribosome that makes it misbehave?' To answer this, we will purify ribosomes caught in the act of translating through the frameshift signal and study them by microscopy. The microscopy technique we will use is based on electrons rather than light, so is extremely powerful, and is called cryo-electron microscopy. It is 'cryo' because the samples are frozen in aqueous solution before examination to keep them in a natural and stable state. The images we obtain will hopefully tell us something about how the frameshift signal interferes with the ribosome, and should also be informative about how ribosomes work during normal protein synthesis.
Technical Summary
Many viral and some cellular mRNAs contain programmed /1 ribosomal frameshifting signals that instruct the ribosome to change reading frame at a defined point and to continue translation. Frameshift signals are often associated with the expression of virus replicases, for example, retroviral reverse transcriptases. The mRNA signal that specifies frameshifting has two components, a slippery sequence of nucleotides, where the frameshift takes place and an essential stimulatory RNA structure (sometimes a stem-loop but more often an RNA pseudoknot). The mechanism of frameshifting is not fully understood, but likely involves a direct interaction between the stimulatory RNA and the ribosome that perturbs the elongation cycle at the time that the slippery sequence is being decoded. Recently, we have succeeded in isolating highly-purified rabbit reticulocyte ribosomes stalled in the act of translating the stimulatory RNA pseudoknot of a coronavirus frameshift signal. In this project, we will use cryo-EM techniques to determine the structure of the stalled 80S-pseudoknot complexes. We will also investigate the structure of ribosomes paused at other stimulatory RNAs, both functional and non-functional in frameshifting. Key macromolecular interactions identified from the cryo-EM reconstructions will be verified by biochemical analysis. The project is a joint venture between a group at Cambridge with experience in the biochemical analysis of ribosomal frameshifting and one at Oxford, with expertise in cryo-EM.
People |
ORCID iD |
Robert Gilbert (Principal Investigator) |
Publications
Brierley I
(2007)
Viral RNA pseudoknots: versatile motifs in gene expression and replication.
in Nature reviews. Microbiology
Gilbert RJ
(2008)
Ribosomal acrobatics in post-transcriptional control.
in Biochemical Society transactions
Moran SJ
(2008)
The mechanics of translocation: a molecular "spring-and-ratchet" system.
in Structure (London, England : 1993)
Namy O
(2006)
A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting
in Nature
Description | -That a mechanical model can be developed for -1 frameshifting in which the stability of the frameshift-inducing pseudoknot determines the frameshift frequency. -That translocation can also be understood mechanically as subject to a "Brownian ratchet", on that basis. |
Exploitation Route | Has the potential to be applied as a therapeutic target in disease caused by viruses such as HIV and coronaviruses. |
Sectors | Education |
Description | Our work suggested a simple mechanical model for how ribosomes translating mRNAs are caused to slip backwards 1 nucleotide, a strategy employed in particular by viruses. Several groups around the world have used our findings as the basis for further work, or have interpreted their data on the basis of our work, see: Hansen et al., PNAS 104, 5830-5835 (2007) Chen et al., PNAS 106, 12706-12711 (2009) Qu et al., Nature 475, 118-121 (2011) Tholstrup et al., Nucleic Acids Rec 40, 303-313 (2012) Ritchie et al., PNAS 109, 16167-16172 (2012) Mouzakis et al., Nucleic Acids Res 41, 1901-1913 (2013) Our work suggested a simple mechanical model for how ribosomes translating mRNAs are caused to slip backwards 1 nucleotide, a strategy employed in particular by viruses, and other groups have used our model to interpret their data on this. |
First Year Of Impact | 2006 |
Sector | Education |
Impact Types | Cultural |
Description | Association of Resources for Biophysical Research in Europe |
Geographic Reach | Europe |
Policy Influence Type | Influenced training of practitioners or researchers |
Impact | We are implementing Europe-wide projects for quality control of biophysical methods and benchmarking of new technologies. Our role builds directly on biophysical and structural biology studies carried out in the lab over several years. It has been strengthened by additional funding which began since our involvement with ARBRE. |
URL | https://www.structuralbiology.eu/networks/association-resources-biophysical-research-europe |
Description | Molecular Biophysics in Europe (MOBIEU) |
Geographic Reach | Europe |
Policy Influence Type | Influenced training of practitioners or researchers |
Impact | MOBIEU is a COST Action which will enable the improvement of biophysical methods development across Europe and with international impact. I am one of two UK members of its Management Committee, nominated through the Department for Business, Information and Skills. |
URL | http://www.cost.eu/COST_Actions/ca/CA15126 |
Description | BBSRC Responsive Mode Project Grant |
Amount | £481,717 (GBP) |
Funding ID | BB.G008051.1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2009 |
End | 03/2013 |
Title | WellMAP |
Description | A new computer program allowing rapid manipulation and assessment of 3D electron density maps. |
Type Of Material | Technology assay or reagent |
Provided To Others? | No |
Impact | It has greatly improved the efficiency of our data analysis. |
Title | WellMAP |
Description | A newly encoded set of operations including novel computational tools for 3D map quality assessment and manipulation. |
Type Of Material | Computer model/algorithm |
Provided To Others? | No |
Impact | It has greatly increased the efficiency of our data analysis. |
Description | The mechanical basis of -1 frameshifting |
Organisation | University of Cambridge |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We collected cryo-EM image data and determined 3D reconstructions of the imaged ribosomal complexes. |
Collaborator Contribution | Dr Ian Brierley's team in Cambridge provided -1 frameshifting complexes for our analysis by structural methods. This funding strengthened our work. Publications from it are still pending. |
Impact | This is multidisplinary - we are biophysicists and structural biologists, Dr Brierley is a virologist and molecular biologist. Outputs are captured in our several papers. |
Title | WellMAP |
Description | WellMAP encodes a set of operations some of which are standard in EM data analysis (eg Fourier shell correlation) and some of which are novel (the Fourier plate correlation) while still others are known elsewhere but were independently coded (B factor calculation and use for data filtration). |
Type Of Technology | Software |
Year Produced | 2011 |
Open Source License? | Yes |
Impact | We have found the software has greatly improved our efficiency of data analysis. |
Description | Media interest (frameshifting mechanism discovery) |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | My collaborator Dr Brierley was interviewed for national radio; I was interviewed for local radio; our work appeared on the BBC website. The media interest highlighted the impact that basic research can have in identifying novel mechanistic models for biological processes. |
Year(s) Of Engagement Activity | 2006 |
URL | http://news.bbc.co.uk/1/hi/health/4757363.stm |
Description | School visit (to Wellcome Trust Centre) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | The talks revealed the value of a structural understanding of biological processes. There seemed a heightened awareness of the value of our work. |
Year(s) Of Engagement Activity | 2012,2013 |