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Access Type

WSU Access

Date of Award

January 2023

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Athar Ansari

Abstract

The central dogma of biology sets forth a foundation for the storage, transmission, and expression of genetic information of a biological organism. Within this doctrine lies fundamental processes which are essential for growth, function, and reproduction of biological organisms. During the first transition in the dogma, from one molecule of DNA to RNA, transcription occurs in a highly regulated and complex fashion. In order to fully understand gene expression, the process of transcription is of utmost importance to scrutinize. The process of transcription is an essential biological event in which RNA is produced from the DNA template, a necessary step in the eventual production of proteins. At the center of the transcription process is the RNA polymerase, an enzyme responsible for catalyzing RNA synthesis. There are many factors involved in transcription, one of which is TFIIB, that is crucial for viability and is canonically involved in the beginning initiation step. TFIIB has normally been considered to be solely involved in initiation however, research from this dissertation demonstrates TFIIB to be involved in many aspects of transcription. To investigate the broader role of TFIIB in transcription, the mechanism by which TFIIB effects transcription in a non-canonical manner was examined. TFIIB was found to copurify with the termination factor Pcf11, only in a chromatin context. Additionally, I performed quantitative proteomic analysis of yeast TFIIB. We purified TFIIB from soluble cell lysate and the chromatin fraction. TFIIB purified from the chromatin exhibits a number of interactions that explain its non-canonical roles in transcription. Apart from preinitiation components RNAPII, TFIIF and TFIIH, all three 3’ end processing-termination complexes; CF1, CPF and Rat1, are significantly enriched in the chromatin-TFIIB preparation. These results explain the presence of TFIIB at the 3’ end of genes, its role in gene looping, and its newly identified role in termination of transcription. The presence of the Lsm complex as well as TREX complex subunit Sub2 in chromatin-TFIIB opens up the possibility of novel roles of TFIIB in synthesis-decay coupling and nucleocytoplasmic transport of mRNA. This multiplicity of functions may contribute to the preferential targeting of TFIIB during viral pathogenesis. Apart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been now been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remains largely unexplored. To determine the prevalence of TFIIB termination role, we performed GRO-Seq analyses in sua7-1 mutant (TFIIBsua7-1) and the isogenic wild type (TFIIBWT) strains of yeast. Almost a three-fold increase in readthrough of the poly(A)-termination signal was observed in TFIIBsua7-1 mutant compared to the TFIIBWT cells. Of all genes analyzed in this study, nearly 74% genes exhibited a statistically significant increase in terminator readthrough in the mutant. To gain an understanding of the mechanistic basis of TFIIB involvement in termination, we performed mass spectrometry of TFIIB, affinity purified from chromatin and soluble cellular fractions, from TFIIBsua7-1 and TFIIBWT cells. TFIIB purified from the chromatin fraction of TFIIBWT cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with both CF1A and Rat1 termination complexes in TFIIBsua7-1 mutant. ChIP assay revealed that the recruitment of Pta1 subunit of CPF complex, Rna15 subunit of CF1 complex and Rat1 subunit of Rat1 complex registered a 25-50% decline in the mutant over wild type cells. Overall conclusion of these results is that TFIIB-termination factor interaction facilitates termination of transcription on a genomewide scale in budding yeast. Transcription by RNAPII can occur bidirectionally, either in the sense or upstream antisense direction. Promoter directionality is a natural phenomenon in which two separate pre-initiation complexes form around a gene’s promoter region, inducing transcription in two directions: sense and upstream antisense. TFIIB is an essential initiation factor however, it has been shown to have non-canonical roles in transcription such as involvement in gene looping and termination. In this study, TFIIB is shown to play a role in the balance of upstream antisense transcription by affecting promoter directionality. For multiple genes examined, production of upstream antisense RNA (uaRNA) is increased compared to mRNA production upon mutation of TFIIB. In excess of gene specific analysis, this promoter directionality defect will be examined on a genomewide scale through the Global Run-On sequencing (GRO-seq) approach. The results presented here demonstrate that TFIIB is involved in enhancing promoter directionality of analyzed genes, as increased uaRNA is produced, resulting in a promoter directionality defect in the TFIIB mutant.

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