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Regulation of transcription initation and elongation by sequence specific DNA binding factors

Akhtar, Asifa; (1997) Regulation of transcription initation and elongation by sequence specific DNA binding factors. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Research described in the first part of this thesis was focused on studying the control of post-transcriptional initiation events in yeast. A transcriptional run-on assay in permeabilised yeast cells was used to study the distribution of RNA polymerase II (Pol II) complexes before and after activation of GAL1 gene by the Gal4 activator. Polymerases were found engaged on the template at the 5' end before activation, but only appeared at the 3' end after activation. Mutations of the Pol II CTD, the CTD kinase Kin28, and the holoenzyme subunit, Srb2, all inhibited formation of 3' polymerases in response to an activator. However, these mutations did not inhibit establishment of polymerases at the 5' end. The differences between 3' and 5' Pol II ternary complexes suggest that they represent qualitatively distinct "activated" and "non-activated" forms of polymerase. The results implicate CTD phosphorylation in a switch from "non-activated" transcription, that is confined to the 5' end, to an "activated" mode that traverses the length of the gene. Second part of the thesis was focused on studying transcriptional activation of c-fos mediated by the serum response element (SRE). A ternary complex with the transcription factors SRF (serum response factor) and TCP (ternary complex factor) is formed at the SRE. Functional RhoA is required for serum or LPA activated transcription at the SRE in a SRF dependent TCF independent manner. In order to isolate factors that directly target SRF in response to RhoA, two yeast screens were performed. The first screen used SRF bound at the SRE, and resulted in isolation of Zhx 1, a protein containing 5 homeoboxes and 2 zinc fingers. Results show that the SRF DNA binding domain is sufficient for interaction with Zhx1. Moreover, stable complexes with SRF and Zhx266-620 can be detected in vitro. However, Zhx1 does not activate SRE containing reporters in a mammalian system. The second screen used SRF bound at the Gal4 operator. This screen resulted in isolation of Sp4, a protein containing 3 zinc fingers. Results show that Sp4312-784 can interact with full length SRF but the SRF DNA binding domain is not sufficient for this interaction. Sp4312-784 can also cause a modest decrease in transcriptional activation of SRE containing reporters in mammalian cells.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Regulation of transcription initation and elongation by sequence specific DNA binding factors
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10099712
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