Date of Award

Fall 2014

Document Type


Degree Name

Doctor of Philosophy (PhD)


Biological Sciences

First Advisor

Karrer, Kathleen

Second Advisor

Schläppi, Michael

Third Advisor

Maurice, Martin St.


RNA processing and turnover plays an important role in RNA maturation, metabolism and quality control, which contribute to gene regulation and cell health. The TRAMP complex, composed of an RNA binding protein Air2p, a poly-A polymerase Trf4p, and a RNA helicase Mtr4p, assist nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae, like tRNAiMet turnover, 5'ETS degradation and rRNA processing. Mtr4p structure reveals a novel protruding arch domain, which contains the recognizable KOW domain and a stalk domain. Except for the two conserved RecA-like helicase domains, the helicase core contains two other structural domains, a winged helix that connects arch to the core and a ratchet domain of unknown function. The role of how structural domains assist Mtr4p function remains unclear. In this study, I created a library of Mtr4p structural domain mutants that were defective in tRNAiMet turnover. KOW domain mutations K700N, P731S, S672N and P802S, winged helix mutation K904N, and ratchet domain mutation R1030G were characterized in cells and biochemically. Mtr4-904p and Mtr4-1030p showed significant defects in tRNAiMet turnover in vivo, while the arch domain mutants didn't cause detectable growth phenotypes. Consistently, Mtr4-904p and Mtr4-1030p exhibited decreased in vitro unwinding activities. However, the modest unwinding defect of K700N was not reflected in cell. It suggests that structural domains in helicase core are more essential for Mtr4p function than that of the arch domain. KOW domain and ratchet domain contributed to RNA binding activities of Mtr4p in vitro. Winged helix mutant K904N had modest effects on RNA binding in vitro, but showed greater effects on protein stability. Structural domain mutants affected ATPase activities. K700N and R1030G mutations reduced Mtr4p ATP affinity. Overall, the results revealed that residues in structural domains affect Mtr4p enzymatic activities from different aspects. In addition, I showed that single-stranded RNA is a preferred substrate for Mtr4p binding, and activates higher enzymatic activity of Mtr4p. My results also demonstrate that, as a helicase, Mtr4p is able to modulate the balance between unwinding and re-annealing in a concentration and ATP dependent manner.