RNR Polymerase I (RNA Pol I) is a “factory” that orchestrate the transcription of ribosomal rRNA for constructing ribosomes as a primary workshop for protein translation to sustain cell growth. Misregulation of RNA Pol I can cause uncontrolled cell proliferation, which leads to the development of cancer. Yeast (Saccharomyces cerevisiae) is a valuable model system to study RNA Pol I. Recently, the X-ray crystal structure of the yeast homologue of RNA Pol I was elucidated, offering the structural basis to selectively target this transcriptional machinery. The approach to selective RNA Pol I targeting was to disrupt the interaction between a specific transcription factor, RRN3 that bind distinct regions of RNA Pol I. For this purpose, a recombined plasmid was designed to carry human rDNA plus its promoter as target together with a selection marker gene. Therefore, this plasmid could not only introduce the target gene into the yeast (host), but also facilitate the passage of this target gene into a stable yeast strain. In this project, one uracil deficient yeast strain of YBR140C was transformed with the recombined yeast integrative plasmid of pHmrDNA-YIPlac211-TG1. This is a recombined plasmid containing not only the human rDNA but also the URA3 gene as a selection marker. PCR amplification of the human ribosomal DNA was indicative of successful integration of the human ribosomal DNA into the genome of the two yeast strains. Virtual screening using a library of 700 FDA-approved compounds was docked into the RRN3-RNA Pol I complex to identify small molecule disruptors of the RRN3-RNA Pol I as a selective strategy. Using growth assays, gel electrophoresis and transcriptional assays, we identified cerivastatin sodium as a lead virtual hit. The result implicates cerivastatin sodium as a selective RNA Pol I inhibitor worthy of further development with potential as targeted anticancer therapeutic.
| Identifer | oai:union.ndltd.org:uky.edu/oai:uknowledge.uky.edu:chemistry_etds-1117 |
| Date | 01 January 2019 |
| Creators | Tan, Xiao |
| Publisher | UKnowledge |
| Source Sets | University of Kentucky |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations--Chemistry |
Page generated in 0.0019 seconds