Mutant Analysis of the RNA Polymerase II Carboxy-terminal Domain

Open Access
Author:
Mccarl, Lauren Hope
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • David Scott Gilmour, Thesis Supervisor
  • Joseph C. Reese, Honors Advisor
Keywords:
  • RNA Polymerase II
  • CTD
  • Drosophila
  • beta-galactosidase assay
  • qRT-PCR
  • transcription
  • mutant analysis
Abstract:
The carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNAPII), is a tail-like extension that enables RNAPII to interact with a myriad of factors essential for transcription and co-transcriptional RNA processing. It is characterized by tandem heptad repeats, the consensus sequence of which is YSPTSPS. Different combinations of phosphorylated residues have been found to correlate with different stages of the transcription cycle. To enhance our understanding of the functionally enigmatic nonconsensus heptads, two distinct generations of CTD mutant transgenic fly lines were generated by the Gilmour lab. The first generation expressed both endogenous RNAPII and a CTD mutant version of the polymerase. The second generation, designed to determine rescue ability, co-expressed short hairpin RNA against endogenous Rpb1 mRNA (shRpb1) and a shRpb1-resistant version of CTD mutant RNAPII. In addition to helping construct several plasmids to create these CTD mutants, I also characterized previously generated mutants by beta-galactosidase (β-gal) staining assay and quantitative real-time PCR (qRT-PCR). The ΔHep(exp) mutant, characterized by the deletion of a highly conserved, eight-repeat region containing the only two conserved heptads of the Drosophila CTD, was of particular interest because it is lethal in adult flies. My β-gal assays and qRT-PCR analysis showed the ΔHep(exp) mutation had largely no effect on hsp70 reporter gene expression or endogenous hsp70 mRNA synthesis, respectively. This suggests the defect associated with ΔHep(exp) may involve a gene (or genes) other than hsp70.