Composition heterogeneity and dynamics of the eukaryotic translation machinery

Lead Research Organisation: University of Manchester
Department Name: Chem Eng and Analytical Science


Many processes in the cell are carried out by large machines, built by the cell from complex assortments of parts. If we encountered a new machine how would we understand it? First, we'd work out what it does, and then, examine the parts that it is made of, and finally, we'd try and reassemble or build our own machines from an equivalent set of parts. If we then wanted to build more machines, we would have to design a factory to make sure that all the parts were available. The design of any manufacturing process is complex - do we for example, make sure that N sets of parts are available if we know we are to build N machines ('just in time' principles) - what if the supplier of one of the parts fails to deliver? Or, do we design a factory where we have 50N parts always available, increasing storage and manufacturing costs (surplus stock). Do we recycle those surplus parts, or store them for years, so that they gradually become damaged or unfit for purpose? The cell faces exactly the same challenges. The machine that makes proteins in the cell is called the ribosome, which uses the blueprints in the genes to dictate the synthesis of proteins. The ribosome is itself a highly complex machine, made of proteins and RNA. Our goal is to characterise all the proteins associated with the translation machinery, calculate how many of each part (protein) there are, and work out how the cell balances the costs and benefits of 'just in time' against 'surplus part' manufacturing. We will discover how rapidly the parts can be made, the degree to which surplus parts are recycled, and even whether the cell makes variants of the machine (S, GT, GTL, GTE models!) that are designed for different uses and locations. By understanding how such complex machinery is made, we will begin to understand how the cell balances flexibility of response (in time, and in terms of types of machine) with quality control, manufacturing principles and energy costs. Manufacturing is as complex on the cellular scale as it is in a car assembly plant.

Technical Summary

Given the fundamental importance of protein synthesis to living systems, we feel that the full characterisation of the macromolecular machinery responsible for this process in eukaryotes should be a high priority. Recent evidence points to the existence of many previously unidentified proteins that either modulate the activity of the translational machinery or play core functional roles. Using a range of techniques, including newly developed mass spectrometric methods, bioinformatics, fluorescence imaging, in vivo functional analysis and in vitro reconstitution, we will perform comprehensive and absolutely quantitative characterisation of the translational machinery and its associated proteins in Saccharomyces cerevisiae. Biophysical and biochemical characterisation will be combined with functional analysis in order to provide a much enhanced picture of the composition, function and control of the translation machinery. Our focus will be on defining the heterogeneity of the system and characterising the changes in its composition in response to environmental changes. Additionally, detailed work on protein component exchange and degradation will elucidate important details of the dynamics and turnover of the translation machinery components.


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Description We have used internally calibrated quantitative mass spectrometry to characterise the ribosome-associated proteins in yeast. This served to determine what additional proteins are associated with the protein synthesis machinery. We focused in particular on the association of the mRNA-decapping activators/translational repressors Dhh1 and Pat1 with the ribosomal fractions obtained by sucrose density gradient centrifugation. Remarkably, both of these proteins are at least partially excluded from polynomial mRNP complexes during exponential growth, and only gain full access under stress conditions, whereupon we suspect they act to inhibit translation. This means that the cell is able to control the localisation of potent translational regulators to match the changing demands for protein synthesis as cells enter stress. We have also found evidence that the mRNP remodelling involved in this process is facilitated by the DEAD-box helicases Ded1 and eIF4A.
Exploitation Route Further work on the technical challenges of determining the functional roles of ribosome-associated proteins would lead to a better definition of the members of the 'translatosome'.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology