Regulatory Studies of the Mammalian RNA Polymerase III Transcriptional Apparatus

Allison, Simon J (2001) Regulatory Studies of the Mammalian RNA Polymerase III Transcriptional Apparatus. PhD thesis, University of Glasgow.

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Although many studies have shown that pol III transcription is strongly regulated in higher eukaryotes, it is poorly understood how this regulation is achieved. The basal pol III factors TFIIIB and TFIIIC have been implicated as common targets for regulation. I have developed reproducible purification protocols for yielding partially purified active human TFIIIB and human TFIIIC. The purity of hTFIIIB and hTFIIIC obtained are a significant improvement upon that of hTFIIIB and hTFIIIC typically used in our laboratory, allowing regulatory studies to be conducted with a much higher level of confidence than previously. One established repressor of pol III transcription is the tumour suppressor RB. Recently, the related proteins pi07 and pi30 have also been shown to inhibit pol III transcription. Here, I show that endogenous p107 and p130 cofractionate and coimmunoprecipitate with endogenous TFIIIB, suggesting that, like RB, p107 and p130 stably associate with TFIIIB under physiological conditions. I have also investigated why the binding of RB to TFIIIB inhibits pol III transcription. For several genes transcribed by pol II, RB represses transcription through the recruitment of the histone deacetylase HDAC1, which is thought to deacetylate histones at the promoter resulting in the formation of a more compact chromatin structure less accessible to transcription factors. However, the repression of pol III transcription in vitro by RB is unaffected by the presence of the histone deacetylase inhibitor trichostatin A. Using an immunoisolated pol III complex that contains pol III, TFIIIC and TFIIIB, I show that recombinant RB can specifically disrupt the interaction between TFIIIB and TFIIIC. The serine/threonine kinase CKII is identified as a novel activator of mammalian pol III transcription and is shown to stably interact with endogenous hTFIIIB. Significantly, CKII kinase activity appears to promote the binding of TFIIIB to TFIIIC. The receptor tyrosine kinase neu (erbB2) is also implicated in the regulation of pol III transcription. A rodent ovarian epithelial cell line transformed by an activated neu oncogene is found to display elevated pol III activity. TFIIIC2 B-block binding activity is specifically elevated. Using the purified TFIIIB and TFIIIC fractions, I show that TFIIIC is, limiting in the untransformed control cell line, indicating that upregulation of TFIIIC2 activity in response to neu transformation can at least partly account for the increase in pol III activity.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Bob White
Keywords: Biochemistry
Date of Award: 2001
Depositing User: Enlighten Team
Unique ID: glathesis:2001-74961
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 14:59
Last Modified: 27 Sep 2019 14:59

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