The Mechanism of NELF Recruitment by DSIF in Promoter-proximal Pausing of RNA Polymerase II
Open Access
- Author:
- Deng, Eilene
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- David Scott Gilmour, Thesis Supervisor
Lorraine C Santy, Thesis Honors Advisor - Keywords:
- promoter-proximal pausing
transcription factors
gene regulation
DSIF
NELF
RNA Polymerase II - Abstract:
- Gene expression is regulated at the transcriptional level, and dysregulation can result in disease. A key regulatory step in early transcription is promoter-proximal pausing of RNA polymerase II (Pol II). On most protein-coding genes in higher eukaryotes (including humans and fruit flies), Pol II initiates transcription and then pauses after transcribing 30-50 nucleotides. This process allows cells to efficiently fine-tune gene expression in response to various signals because the pause duration controls the level of expression. Two proteins, DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), promote pausing by cooperatively binding to Pol II. DSIF is composed of subunits Spt4 and Spt5, while NELF is composed of subunits NELF-A, -B, -D, and -E. While DSIF can bind to Pol II without NELF, NELF binding requires the presence of DSIF in a transcriptionally-active Pol II-DSIF complex. The molecular basis underlying this dependency remains unknown. I used Drosophila melanogaster as a model system to investigate the mechanism of NELF recruitment. I replaced domains of D. melanogaster DSIF with homologous domains from C. elegans and S. cerevisiae, neither of which have NELF and therefore would likely not have a NELF-binding domain. I generated ten DSIF mutants based on previously published structural data. The mutants were recombinantly expressed in E. coli and purified using affinity chromatography. I successfully purified several Spt5 mutants in which the KOW2-3 or KOW4 domains were substituted. However, for Spt4 mutants in which I substituted either a region or the entirety of Spt4, Spt4 dissociated from Spt5 due to disruption of key protein interfaces. Using a gel shift assay, the Spt5 mutants were confirmed to bind to Pol II, indicating that the mutations do not disrupt Pol II-DSIF binding. The mutants were then screened for NELF binding using a gel shift assay. The mutant in which the S. cerevisiae sequence was placed in the KOW2-3 domain of Spt5 showed reduced NELF binding, suggesting that the KOW2-3 domain may play a role in NELF recruitment. Several mutants in which the KOW4 domain was mutated did not show impaired NELF binding, suggesting that the KOW4 domain is not involved in NELF recruitment. This study provides critical insight into the mechanism of promoter-proximal pausing.