Structural and functional analysis of ribosome initiation and ribosomal frameshifting.
Lead Research Organisation:
University of Cambridge
Department Name: Pathology
Abstract
Proteins are polymers of amino acids. The order of the amino acids is governed by the genetic code in the genes of the cell's DNA. In protein synthesis, a gene is first copied into messenger RNA (mRNA) - a single-strand copy of the DNA which retains the code - and then taken to the ribosome. Ribosomes are protein synthesising machines some 25-35nm in diameter and made up of two subunits, one large, one small. The mRNA is loaded linearly between the subunits, whereupon adaptor molecules called transfer RNAs (tRNAs) arrive to decode the mRNA. Each transfer RNA has an amino acid at one end, and at the other, a region which recognises a specific run of three consecutive nucleotides in the mRNA code, a 'triplet'. During protein synthesis, the ribosome moves along the mRNA triplet by triplet and at each step, a tRNA with the appropriate recognition sequence is recruited and the ribosome transfers the tRNA's amino acid to the growing chain of amino acids, the polypeptide. This project will study three aspects of protein synthesis. The first is how mRNA is recruited to the ribosome and fed between the subunits. In mammals, the mRNA interacts with the isolated small subunit of the ribosome in complex with a host of other proteins termed initiation factors. This initiation complex moves along the mRNA, scanning the sequence until the specific triplet that signifies the start of protein synthesis is identified. At this point, the large subunit joins and amino acid polymerisation begins. We wish to characterise in detail the structure of this scanning complex (termed the 48S complex), by purifying initiation complexes paused in the act of scanning along the mRNA, or at the initiation triplet, and study them using microscopy. Our microscope uses electrons rather than light, and is extremely powerful because the short wavelength of electrons allows the visualisation of fine molecular detail. The samples are also frozen at -180C, which keeps them in a natural and stable state, and so the technique is known as 'cryo-electron microscopy' (cryo-EM). The images we obtain will show us the structure of the 48S complex and tell us something of how the various initiation factors allow the small subunit to scan along the mRNA and to start protein synthesis. The second aspect is elongation, when amino acids are added, via tRNAs, to the growing chain. Inside the ribosome are multiple binding sites for tRNAs. A tRNA comes in at one site, binds to its complementary mRNA triplet and transfers its amino acid to the growing chain, moves to an adjacent site to make space for the next tRNA, and finally leaves the ribosome via an exit site. The movement of tRNAs has been hard to analyse at a molecular level. We have identified an mRNA signal termed a pseudoknot which blocks the progress of the ribosome along the mRNA and stalls the ribosome at the point when the tRNAs are moving within the ribosome. We will use cryo-EM to study the structure of ribosomes stalled at a pseudoknot to elucidate the molecular details of tRNA movement. Pseudoknots can also cause the ribosome to change reading frame, that is, to shift from reading one set of triplets (one frame) to reading an overlapping set (another frame), and this leads to a completely new amino acid sequence in the protein. Cryo-EM studies of pseudoknot-stalled ribosomes may thus be informative as to how the ribosome maintains the correct reading frame. The final aspect of the project concerns the structure of ribosomes prepared from mammalian cells. Because the ribosome plays a major role in controlling the rate of protein synthesis, and even which proteins are made, in different cell states the ribosome becomes modified in a variety of ways that affect which proteins are made and how rapidly. By determining structures for ribosomes from cells grown under known conditions we will make the first moves in showing the relationship between ribosome mechanism and regulatory processes in living cells.
Technical Summary
Protein synthesis requires that mRNAs be recruited to the ribosome and processed through it, the genetic code being read into an amino acid sequence. mRNA recruitment involves a series of eukaryotic initiation factors (eIFs) that bind to and modify the architecture of the small ribosomal subunit. Two key complexes are formed during this process: the 43S complex in which the 40S small subunit is bound with several protein initiation factors (eIFs) that open its structure up to bind mRNA, and the mRNA-bound form which has additional eIFs bound and is known as the 48S complex. We aim to purify 48S complexes from mammalian in vitro translation reactions derived from rabbit and human cells and to determine its structure by cryo-electron microscopy (cryo-EM). This will allow us to show how the host of eIFs facilitate mRNA recruitment and scanning to find the AUG start codon. In a parallel project, we will continue our previous work on ribosomes at the polypeptide elongation phase of protein synthesis. The translocation of tRNAs through the ribosomal intersubunit space maintains the triplet codon reading frame. Specific signals in the mRNA itself can cause frameshifting, either -1 or +1, to generate a different protein sequence. Current data indicate a molecular spring-and-ratchet mechanism for translocation of tRNA, confounded in the presence of frameshift-stimulating secondary structure in the mRNA, which generates the reading frame change. We will study pseudoknot and stem-loop folded mRNAs of known structure in the context of the translating ribosome to determine in greater detail how they cause the frameshift to occur. A further study involving 80S ribosomes will look at how protein synthesis is regulated in the cell during the elongation phase of synthesis. This will focus on the modifications accompanying changes in cell growth rate, and how this is reflected in the conformation of the ribosome and its binding by regulatory proteins and other ligands.
People |
ORCID iD |
Ian Brierley (Principal Investigator) |
Publications
Brierley I
(2013)
Macrolide-induced ribosomal frameshifting: a new route to antibiotic resistance.
in Molecular cell
Finch LK
(2015)
Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.
in Journal of virology
Firth AE
(2012)
Non-canonical translation in RNA viruses.
in The Journal of general virology
Flanagan JF
(2010)
Direct observation of distinct A/P hybrid-state tRNAs in translocating ribosomes.
in Structure (London, England : 1993)
Li Y
(2014)
Transactivation of programmed ribosomal frameshifting by a viral protein.
in Proceedings of the National Academy of Sciences of the United States of America
Lin Z
(2012)
Spacer-length dependence of programmed -1 or -2 ribosomal frameshifting on a U6A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting.
in Nucleic acids research
Miyadera K
(2012)
Multiple mechanisms contribute to leakiness of a frameshift mutation in canine cone-rod dystrophy.
in PloS one
Nikolic EI
(2012)
Modulation of ribosomal frameshifting frequency and its effect on the replication of Rous sarcoma virus.
in Journal of virology
Prado JG
(2009)
Functional consequences of human immunodeficiency virus escape from an HLA-B*13-restricted CD8+ T-cell epitope in p1 Gag protein.
in Journal of virology
Strnadova P
(2015)
Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence.
in PLoS pathogens
Description | 1. We determined the cryo-EM structure of two mammalian translocating ribosomal complexes, stalled at a frameshift-promoting RNA pseudoknot, containing distinct A/P hybrid-state tRNAs but only one possessing eEF2. A model of translocation was proposed in which eEF2, in the face of stable mRNA secondary structures, can cycle off from and on to the ribosome for further attempts at translocation. 2. Cryo-EM reconstructions were prepared of 80S rabbit ribosomes stalled at the stimulatory RNAs of a variety of functional viral pseudoknots including HIV-1, BWYV, variants of the IBV pseudoknot with extended loop 3, and a non-functional (in frameshifting) pseudoknot. Reconstructions of a rabbit 48S initiation complexes are underway. 3. Studies of antisense oligonucleotide-mediated frameshifting identified both -1 and -2 frameshifting on a U6A heptamer. Differences in optimal spacing of -1 and -2 frameshifting strongly supports our mechanical model of frameshifting proposed from the cryo-EM work. |
Exploitation Route | EF2 cycling model is now being considered likely by other groups and may be included in future translocation models. |
Sectors | Education |
Description | Wellcome Investigator Award |
Amount | £774,356 (GBP) |
Funding ID | 202797/Z/16/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 11/2016 |
End | 10/2021 |
Title | Computational separation of ligand-bound ribosomal states |
Description | We developed novel tools and ourselves implemented those described by others for the computational separation of different ligand-bound ribosome states. This directly resulted in the paper Flanagan et al Structure 18, 257-264 (2010) and will result in several more papers currently in preparation. We have also described some of the methods developed in a paper currently under review at Ultramicroscopy. Methods developed include omit analysis, B-factor determination for cryo-EM structures, cross-refinement of independent structures and reference-free validation of ligand occupancy. |
Type Of Material | Computer model/algorithm |
Year Produced | 2009 |
Provided To Others? | Yes |
Impact | Similar methods have been adopted by other structural biologists. |
Description | CryoEM analysis of ribosome frameshifting |
Organisation | Medical Research Council (MRC) |
Department | MRC Laboratory of Molecular Biology (LMB) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Using the knowledge gained during the period of this BBSRC grant, we are now attempting to carry out high resolution cryoEM analysis of ribosomes stalled at sites of ribosome frameshifting. We are providing the purified ribosomes. |
Collaborator Contribution | Professor Ramakrishnan and his laboratory are providing the facilities for high resolution cryo EM. |
Impact | NO outcomes as yet. The collaboration is multidisciplinary (biology, physics) |
Start Year | 2017 |
Description | Alternate Recodings II |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Participants in your research and patient groups |
Results and Impact | This was a scientific workshop bringing together participants from diverse biological fields. The aim was to alert scientists to the huge amount of related work that was going on in smaller laboratories that few had appreciated. Several collaborative proposals were made at the meeting and I personally have been contacted for advice on numerous occasions since the workshop. |
Year(s) Of Engagement Activity | 2014 |
URL | http://events.embo.org/14-recoding/ |