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Applications of mass spectrometry to study ribosome structure and function

Reference	BB/D000696/1
Principal Investigator / Supervisor	Professor Carol Robinson
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Chemistry
Funding type	Research
Value (£)	300,878
Status	Completed
Type	Research Grant
Start date	01/11/2005
End date	31/01/2009
Duration	39 months

Abstract

The ribosome is the cellular machinery responsible for translation of the genetic code into protein. The fascinating insights into ribosome structure that have been obtained through high resolution X-ray analysis have prompted many questions into the mechanism of action of these large and heterogeneous particles. In particular the role of dynamic regions of ribosomes in facilitating protein synthesis is not well understood largely because these regions are inaccessible in high resolution X-ray structures as they are heterogeneous and very dynamic. By contrast in MS experiments the very dynamic regions are the most accessible. We have found from previous MS experiments that the so called stalks of the ribosome, which play a vital role in delivering factors, are readily released in the gas phase as intact sub-complexes. Interestingly, despite existing dogma that this complex is pentameric, recent MS evidence suggests that the complex is of variable composition containing different numbers of L7/L12 proteins. We plan to follow up this exciting discovery by investigating a wide range of ribosomes from a variety of microbial origins and to determine if a pattern arises between copy number and other properties of the microbial ribosome or associated proteins. In addition to studies of intact stalks we plan to apply the MS methods that we have developed to study structural changes in ribosomes in a variety of different complexes. Of particular interest is the current controversy surrounding the binding site of a factor which allows ribosomes to be recycled after protein synthesis is completed. Using the loss of proteins from the ribosome as a sensitive probe of protein RNA interaction we will study release of proteins and relate this information to perturbation of protein RNA interactions. One of the remaining unsolved mysteries of the ribosome is the question folding of any segments of the nascent chain can take place in tunnel through which the newly synthesised proteinemerges. We will address this question using established techniques to probe folding within the context of the ribosome assembly. We also aim to extend our studies of prokaryotic ribosomes to those from eukaryotes. Specifically we will investigate interactions of eukaryotic ribosomes with viruses, that have the ability to hijack ribosomes and take over protein synthesis in infected individuals.

Summary

In every living cell proteins are made by large molecular machines, the ribosome, which consist of three large pieces of RNA and over 50 proteins, divided into two subunits. These have been studied for almost 50 years and we now have beautiful pictures of their structures from X-ray crystallography. However, these pictures do not show us the mobile parts of the ribosomes and they are some of the most interesting biological features. Using mass spectrometry (MS) we can look at these mobile parts. MS measures the mass of ions in the gasphase; in our laboratory we can observe ribosomes intact or dissociate them and the elusive mobile feature known as the stalk tends to come off first as an intact entity. We have just made the exciting discovery that the number of proteins in the stalk is not the same in all species of bacteria, as was always thought. We want to investigate this further by looking at more species to establish a pattern. Through our knowledge of how ribosomes dissociate we are also able to draw conclusions about changes in the folding and overall shape of the ribosome from our data, especially on binding of other proteins or molecules. We want to use this to study dynamic processes that make up the activity of the ribosome, such as the start (initiation) and end (termination) of protein synthesis, which involves binding various factors, folding of the protein while it is being made (the nascent chain) and still inside the ribosome. We also want to investigate a possible interaction with another cellular machine, which destroys ribonucleic acid, known as the degradosome. Finally, we want to use MS as a method to select and prepare ribosomes for study by Electron Microscopy. This is a very ambitious goal for us but one that we believe will be very exciting.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
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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