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Defining novel complexes involved in translation - a mass spectrometry approach involving TAP-tag isolation and analysis of intact complexes

Reference	BB/C005295/1
Principal Investigator / Supervisor	Professor Carol Robinson
Co-Investigators / Co-Supervisors	Dr Helena Hernandez
Institution	University of Cambridge
Department	Chemistry
Funding type	Research
Value (£)	435,863
Status	Completed
Type	Research Grant
Start date	01/04/2005
End date	31/03/2008
Duration	36 months

Abstract

It is clear that the increased complexity of higher organisms derives from a complicated network of protein interactions, either stable or transient, within the cell. The description of protein interactions is therefore a major research goal in the post-genomic era. The principal aims of this proposal are to develop mass spectrometry methods of describing both transient and stable macromolecular complexes as well as to gain structural insight into the biological roles of protein and RNA-protein complexes in pre-mRNA splicing and ribosome biogenesis. We have established collaboration with scientists with expertise in isolation of native complexes (Bertand Seraphin) and bioinformatics (Sarah Teichmann) to achieve this goal. We believe that it is timely to marry these methods to generate novel generic strategies for describing protein interactions in whole complexes. The tandem affinity purification (TAPtag) method developed by Seraphin is already proven as a powerful method for rapid isolation and characterisation of the protein components of RNA-protein complexes in yeast. In this proposal we aim to develop this method further such that intact complexes can be verified enabling us to establish the copy number of the proteins present, the RNA component as well as the metal binding properties of the proteins. Previously we have shown that it is possible to maintain intact megaDalton complexes in the mass spectrometer that are both unstable and heterogeneous and that both in-source dissociation and MS/MS experiments provide insight into stoichiometry as well as subsets of interacting components within the complex. We will focus initially on the complexes involved in pre-mRNA splicing. The spliceosome consists of large RNA-protein complexes (snRNPs) and other protein components that assemble transiently on pre-mRNA substrates and therefore provide an ideal system for developing our strategies. The generic strategies that we develop using this model system will then be applied to ribosome biogenesis and macromolecular complexes involved in other cellular processes.

Summary

unavailable

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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