The regulation of pol III transcription by mTOR

Graham, Emma Louise (2005) The regulation of pol III transcription by mTOR. PhD thesis, University of Glasgow.

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Abstract

Regulation of protein synthesis is an important aspect of growth control. A major determinant of this process is the availability of tRNA and 5S rRNA, which are synthesised by RNA Polymerase (pol) III. Pol III transcription is tightly linked to growth conditions, decreasing when nutrients or serum factors are low and increasing upon mitogenic stimulation. Therefore, it seems inevitable that mechanisms have evolved to couple production of the biosynthetic machinery with the needs of the cell. The target of rapamycin (TOR) signalling pathway, in conjunction with signalling through the phosphoinositide 3-kinase (PI3K) pathway, is central to this process in a diverse number of organisms. Mammalian cell lines were investigated to assess if pol III transcription is under the control of the PI3K and mammalian TOR (mTOR) pathways. Levels of pol III transcripts were reduced in vivo and pol III transcription was reduced in vitro in response to inhibition of the pathways. Inhibition of the mTOR and PI3K pathways was not found to alter the abundance of the pol Ill-specific transcription factors TFIIIB and TFIIIC, or indeed of pol III itself. Moreover, the effects of inhibition of the pathways on pol III transcription were found to be independent of known regulators of pol III, such as c-Myc, Retinoblastoma protein (RB) or extracellular signal-regulated kinase (ERK) signalling, but were shown to be due, in part, to signalling through the translational effector kinase S6K1. When the mTOR pathway is blocked by rapamycin, the interactions between TFIIIB and TFIIIIC, and between TFIIIB and pol III are ablated, which correlates with the finding that mTOR activity is required for normal promoter occupancy at pol III promoters. These data may be explained by the finding that the mTOR pathway regulates phosphorylation of TFIIIB and TFIIIC subunits. Maf1, a known negative regulator of pol III transcription in yeast, was investigated, since it has been reported to be under the control of the TOR signalling pathway. Maf1 was found to repress transcription of all class III genes in mammalian cells, and this repression can be relieved by the addition of a purified fraction of TFIIIB. Direct interaction of Maf1 with pol III and with the Brf1 subunit of TFIIIB was demonstrated, and further investigation shows that Maf1, pol III and Brf1 follow the same pattern of promoter occupancy on tRNALeu" genes in response to stress. In vivo phospho-labelling and in vitro kinase assays demonstrated that Maf1 is a phosphoprotein, and the phosphorylation of Maf1 was found to be inhibited by both serum-starvation and rapamycin-treatment of cells. This suggests that Maf1 may receive signals from these signalling pathways to co-ordinate pol III activity, and hence the growth capacity of the cell, with nutrient availability.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Pamela Scott
Keywords: Biochemistry
Date of Award: 2005
Depositing User: Enlighten Team
Unique ID: glathesis:2005-71450
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 14:38
Last Modified: 10 May 2019 14:38
URI: http://theses.gla.ac.uk/id/eprint/71450

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