A Genetic and Biochemical Analysis of the Role of the Ccr4-not Complex in Transcription Elongation and Recovery from DNA Damage in Budding Yeast

Open Access
Author:
Libert, Diane Marie
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Joseph C. Reese, Thesis Supervisor
  • Chen Pei David Tu, Honors Advisor
  • Richard John Frisque, Faculty Reader
Keywords:
  • RNA Polymerase II
  • Transcription Elongation
  • Transcription-coupled repair
  • Ccr4-Not
  • Not4
  • E3 Ubiquitin Ligase
  • RING domain
Abstract:
Cells require proper gene transcription in order to produce functional proteins. DNA damage, one obstacle to transcription elongation by RNA Polymerase II (RNAPII), is repaired through pathways such as transcription-coupled repair (TCR). TCR begins when cell machinery senses that RNAPII has stalled on a gene. Before DNA-repair proteins act on the mutation site, RNAPII must be removed, perhaps through ubiquitin-dependent proteolysis of Rpb1, its large subunit. Studies in budding yeast S.cerevisiae have shown that the Rpb1 is degraded in response to DNA damage, but the complete mechanism behind RNAPII turnover remains unclear. The multisubunit Ccr4-Not complex is already known to act in transcription regulation via its roles in nuclear processes such as chromatin remodeling, DNA repair, transcription elongation and activation, and RNA processing and regulation. This complex has two major catalytic activities, deadenylation and ubiquitylation. Intrigued by the ubiquitin-ligase activity of Ccr4-Not, we investigate its role in Rpb1 degradation for TCR. Our experiments indicate that Ccr4-Not subunits Ccr4 and Dhh1, which are involved in mRNA turnover, did not have a noticeable impact on Rpb1 degradation. However, Rpb1 levels in Ccr4-Not mutants under DNA-damaging conditions suggest that Not4, a ubiquitin ligase, does play a role in RNAPII turnover. Not4 possesses a Really Interesting New Gene (RING) domain, a zinc-finger motif responsible for the function of many E3 ubiquitin ligases. Our experiments indicate that the Not4 RING domain is responsible for this subunit’s role in Rbp1 turnover under DNA-damaging conditions. Further investigation will verify this and determine whether Not4 plays a direct or indirect part in Rpb1 ubiquitylation and degradation. By clarifying the role of Ccr4-Not in the removal of RNAPII for TCR, our studies will shed light on the manner in which cells handle genomic damage.