mRNA translation is one of the fundamental and well controlled cellular process requiringthe combined function of a large number of molecular components. The three main stagesof translation, initiation, elongation and termination are facilitated by more than 20 proteinsknown as translation factors. Translation is the final step in the flow of genetic information,and regulation at this level allows for an immediate and rapid response to changes inphysiological conditions. The control exerted at the systems level of translation has notprecisely been characterized. Three different techniques have been employed toquantitative the control exerted by the respective translation factors.In the first approach, employing the microscopic techniques, in vivo intra-cellulardistribution of translation elongation and release factors were analysed with TCM and GFPtags. The result indicates that the factors are cytoplasmically distributed which cannotinfluence the overall translational control. In the second approach, the protein expressionlevels of the elongation and release factors were titrated progressively to explore theircontrol effects on global translation regulation. The endogenous promoter of eachtranslation factor was substituted by the tetO7 synthetic promoter to regulate the expressionlevel in response to varying concentrations of doxycycline. Measurement of proteinsynthesis rate and the growth rate at different levels of the elongation and release factorsprovide insight to system-level control. The results indicate that the elongation factorseEF1A and eEF2 and the release factor eRF1 exert an unexpectedly high degree of controlover translation rate. Moreover, these factors, along with elongation factor eEF3 werefound to be functionally dedicated to translation, in contrast to eEF1B and eRF3, which isevidently multifunctional. In the third approach, a mathematical model has been developedto represent the control landscape of the translational machinery. This translation model is apowerful tool that will be used in the quantitative analysis of translation when two factorsare made limiting at a time. The extensive study carried out on the translational regulationof Saccharomyces cerevisiae reveals an interesting observation of the involvement of eachtranslation factors. For the first time, the quantitative measurement of the translationalregulation reveals the translational regulation exerted by individual translation factors.
|Date of Award||1 Aug 2011|
- The University of Manchester
|Supervisor||John Mccarthy (Supervisor) & David Broomhead (Supervisor)|