Identification of Regions in Drosophila Spt5 That Are Involved in Promoter Proximal Pausing
Open Access
- Author:
- Qiu, Yijun
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 27, 2016
- Committee Members:
- David Scott Gilmour, Dissertation Advisor/Co-Advisor
David Scott Gilmour, Committee Chair/Co-Chair
Joseph C. Reese, Committee Member
Benjamin Franklin Pugh, Committee Member
Philip C. Bevilacqua, Outside Member
Song Tan, Committee Member - Keywords:
- transcription elongation
RNA polymerase II
promoter proximal pausing
Spt5
KOW domain
gene regulation - Abstract:
- Promoter proximal pausing has been recognized as a general feature of transcription by RNA polymerase II in metazoans. It serves as an important regulatory step for gene expression, but the underlying mechanism is not well understood. Two factors, DRB-sensitivity inducing factor (DSIF) and negative elongation factor (NELF) have been recognized as central to the establishment of pausing. They associate with Pol II after it has transcribed around 20 nucleotides and cause Pol II to pause in the promoter proximal region. My work has focused on one of these factors, DSIF. The large subunit of DSIF, Spt5 is a universally conserved transcription elongation factor containing multiple domains. How these domains regulate its multiple functions in transcription is not known. In order to identify regions in Spt5 that are involved in pausing, I constructed Spt5 mutant forms of DSIF and tested their capacity to restore promoter proximal pausing to DSIF-depleted Drosophila nuclear extracts. The C-terminal repeats (CTR) region of Spt5, which has been implicated in both inhibition and stimulation of elongation, is dispensable for promoter proximal pausing. A region encompassing the KOW4 and KOW5 domains of Spt5 is essential for pausing. Mutations in this region reduced the affinity of Spt4/5 to Pol II elongation complex. In addition, mutations in the KOW5 domain shift the location of the pause downstream. Protein-RNA crosslinking analyses reveal that KOW5 directly contacts the nascent transcript and that mutations in KOW5 disrupt this interaction. My results suggest that KOW5 influences the location of the pause through its contact with the nascent transcript. The identification and biochemical analysis of functional domains of Spt5 involved in pausing provide information towards understanding how DSIF contributes to the establishment of pausing. Furthermore, I developed a strategy to evaluate the functions of these mutants in vivo and showed that the KOW4-5 mutants that are pausing-defective in vitro also impair the development of flies. This strategy paves the way for genome-wide analysis on how these mutations affect pausing in vivo.