The role of Spt5-nucleic acid contacts in promoter proximal pausing of RNA polymerase II
Restricted (Penn State Only)
- Author:
- Dollinger, Roberta
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 18, 2023
- Committee Members:
- Melissa Rolls, Major Field Member
David Gilmour, Chair & Dissertation Advisor
Scott Showalter, Outside Unit & Field Member
Song Tan, Major Field Member
Katsuhiko Murakami, Major Field Member
Wendy Hanna-Rose, Program Head/Chair - Keywords:
- RNA polymerase II
DSIF
NELF
transcription
promoter proximal pausing
RNA
DNA
NGN
NusG
KOW
elongation factors
Spt5 - Abstract:
- Promoter proximal pausing of RNA Polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires DSIF and NELF. DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. I used a highly purified in vitro system and Drosophila nuclear extract to investigate the role of DSIF in promoter proximal pausing. I identified two domains of Spt5, the KOW4 and NGN domains, that directly facilitate Pol II pausing. The KOW4 domain promotes pausing through its interaction with the nascent RNA while the NGN domain does so through a short alpha helical motif that is in close proximity to the non-transcribed DNA template strand. Mutation of this alpha helical motif in Drosophila has a male-specific dominant negative effect. The alpha helical motif is also needed to support fly viability. I also show that the interaction between the Spt5 KOW1 domain and the upstream DNA helix is required for DSIF association with the Pol II elongation complex and that DSIF-Pol II association is independent of the nascent transcript. Disruption of the KOW1-DNA interaction is dominant lethal in vivo. Finally, I show that the KOW2-3 domain of Spt5 mediates the recruitment of NELF to the elongation complex.