Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Athar Ansari

Abstract

Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, is emerging as an important gene regulatory mechanism in eukaryotes. The role of promoter bound general transcription factors during initiation is well established. However, recent studies have revealed that some initiation factors also interact with the 3' end of a gene. The biological role of initiation factors at the 3' end of a gene is unknown. The general transcription factors TFIIB and TFIIH have been found to interact genetically with Ssu72, a component of CPF 3' end processing complex. Accordingly, we found that TFIIB and TFIIH localize to the distal ends of genes in a transcription dependent manner. TFIIB localization at the terminator region during transcription requires a functional CF1 complex. TFIIB physically interacts with the all subunits of the CF1 complex in an activator dependent manner. TFIIH also interacts with the CF1 and CPF 3' end processing complexes in a manner depending on its kinase activity. Employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the CF1 complex in yeast cells to form a holo-TFIIB complex. This complex was resistant to MNase digestion and brief exposure to high salt. The sedimentation coefficient of the holo-TFIIB complex was intermediate between that of TFIIH and TFIID. Initiation factors which remain bound on a promoter scaffold in vitro, were not found in a holo-TFIIB complex with termination factors. The holo-TFIIB complex was observed only in the looping competent strains, but not in the looping defective sua7-1 strain. We further show that in sua7-1 cells, where a holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results strongly suggest a role for TFIIB in termination of transcription. Similarly, the kinase dependent presence of TFIIH at the 3' end of genes suggested a role for the factor in termination. Accordingly, we show that RNAP II read through the termination signal in the absence of Kin28 kinase activity. Furthermore, the recruitment of CF1 and CPF subunits at the 3' end of a gene is impaired in the TFIIH kinase defective mutant. We propose that initiation factors are in contact with the terminator during gene looping and play an active role in transcription termination.

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