Structural studies of eukaryotic protein synthesis factor complexes by Cryo EM
Lead Research Organisation:
University of Manchester
Department Name: School of Biological Sciences
Abstract
Molecular recognition drives almost all biological reactions. This project aims to uncover structural details of the interactions between protein factors critical for translation in all eukaryotic cells. The specific translation initiation factors are the G protein eIF2 and two regulators called eIF2B and eIF5. eIF2 is required to bind initiator tRNA to ribosomes. Its GTP/GDP status is controlled by eIF2B and eIF5. Phosphorylation of eIF2 at a specific serine residue inhibits eIF2B activity by forming a non-productive eIF2:eIF2B complex. This mechanism of translational control is conserved across all studied eukaryotes and is important within diverse settings including developmental, metabolism and long-term memory formation. eIF2 and eIF2B are both large multi-subunit complexes, and mutations within these factors cause neurological diseases.
We aim to deconstruct the mechanism of action of these complexes, and their regulation processes. The structure and interactions between purified translation factor complexes will be elucidated by state-of-the-art molecular cryo electron microscopy techniques.
This project incorporates cutting-edge biophysical analysis methods with innovative data analysis. It will blend modern precision molecular biology techniques with biochemistry and yeast genetics. The project involves protein expression and purification, molecular biology, electron microscopy, 3D structure analysis and molecular model building.
This project will require 3D visualisation of data and statistical information to reliably interpret small differences in different protein complexes. We aim to get information on protein complex motion and dynamic information, as well as individual structures. Novel visualisation methods will be developed, as well as new quantitative 3D
image analysis methods.
We aim to deconstruct the mechanism of action of these complexes, and their regulation processes. The structure and interactions between purified translation factor complexes will be elucidated by state-of-the-art molecular cryo electron microscopy techniques.
This project incorporates cutting-edge biophysical analysis methods with innovative data analysis. It will blend modern precision molecular biology techniques with biochemistry and yeast genetics. The project involves protein expression and purification, molecular biology, electron microscopy, 3D structure analysis and molecular model building.
This project will require 3D visualisation of data and statistical information to reliably interpret small differences in different protein complexes. We aim to get information on protein complex motion and dynamic information, as well as individual structures. Novel visualisation methods will be developed, as well as new quantitative 3D
image analysis methods.
Organisations
People |
ORCID iD |
| Alan Roseman (Primary Supervisor) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M011208/1 | 01/10/2015 | 31/03/2024 | |||
| 1908705 | Studentship | BB/M011208/1 | 01/10/2017 | 30/12/2021 |
| Description | Eukaryotic cells can adapt to a variety of stress stimuli by activating a signalling pathway called Integrated Stress Response (ISR). One of ISR main events involves eukaryotic Initiation Factor 2 (eIF2), a trimeric protein that plays a key role in the process of protein synthesis (translation). Under stress conditions eIF2 is modified by the addition of a phosphate group on its alpha subunit, which increases eIF2 affinity for its decameric binding partner and activator eIF2B. As a result, the pool of active eIF2 available for the translation cycle is depleted, and the overall level of protein synthesis is attenuated. To shed light on this mechanism of translational control we used cryo-electron microscopy to determine the three-dimensional structure of the eIF2:eIF2B complex both in its unphosphorylated and phosphorylated forms. The structures show two molecules of eIF2 simultaneously binding to eIF2B. In particular, eIF2a interacts with the regulatory core of eIF2B in a highly similar fashion of the phosphorylated eIF2 alpha. This evidence allows us to speculate on the effects of eIF2 modification on eIF2:eIF2B stability, and propose a model for eIF2 activation at each translation cycle. A library of eIF2 and eIF2B mutants has been created to investigate the two binding pockets that eIF2B has for eIF2. Currently, these protein versions are being tested genetically and biochemically to identify the most promising candidates to use for structural studies. |
| Exploitation Route | Our recent data point out at some key residues in eIF2 and eIF2B that mediate protein-protein interaction in different conformations. However, further investigation is needed to fully elucidate the specific function that these amino acids cover in the binding mechanism. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| URL | https://www.nature.com/articles/s41467-019-10167-3 |
| Description | Facilitating Excellence Fund |
| Amount | £3,450 (GBP) |
| Organisation | University of Manchester |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 02/2021 |
| End | 07/2021 |