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Title:Mechanisms of leucyl-tRNA synthetase dependent group I intron splicing
Author(s):Li, Zhongyi
Director of Research:Martinis, Susan A
Doctoral Committee Chair(s):Martinis, Susan A
Doctoral Committee Member(s):Huang, Raven H; Jin, Hong; Kalsotra, Auinash
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):tRNA synthetase
group I intron
splicing
Abstract:Leucyl-tRNA synthetase (LeuRS) plays dual roles within the yeast mitochondria. In addition to protein synthesis, it is also essential to RNA splicing of critical respiratory genes. The LeuRS collaborates with a maturase to excise the bI4 and aI4α introns from the cob and cox1α genes respectively. The LeuRS-based suppressor mutations have been isolated within the amino acid editing CP1 domain and restore native RNA splicing activity in the presence of an inactive maturase. Mutational analysis of these sites and the regions that surround them demonstrated that certain substitutions can also inactivate LeuRS-dependent splicing activity under in vivo and in vitro conditions. Binding measurements suggest that these suppressor sites are important in maintaining interaction between LeuRS and the group I intron RNA. Thus, CP1 domain binds specifically to the bI4 and aI4α intron to promote RNA splicing. In addition to LeuRS from yeast mitochondria (ymLeuRS), diverse LeuRSs from varied origins such as M. tuberculosis and human mitochondria complement the ymLeuRS activities. Similarly, wild-type E. coli LeuRS (EcLeuRS) complemented a ymLeuRS null strain. Interestingly, at reduced levels of EcLeuRS expression in yeast mitochondria, the heterologous synthetase supported protein synthesis, but not intron splicing. Thus, it is a weak splicing suppressor. Surprisingly, a gain of splicing activity was exhibited by positive charge substitutions at the Ala293 position, suggesting that this Ala293 can be adapted for alternative activities. Preliminary footprinting data suggest that LeuRS binds to the P4-P6 core region of the bI4 intron that is cognate to LeuRS. The RNA duplex mimics of the P6 helix were designed and it was shown that LeuRS promotes their annealing in an ATP-independent manner. Domain analysis of LeuRS shows that the C-terminal domain is critical to the RNA annealing activity. Yeast mitochondrial tRNALeu (ymtRNALeu) competitively inhibit annealing. Also, an ymtRNALeu variable-stem-like region was identified on the P6 stem that is important for LeuRS-dependent annealing. These data support that the annealing and tRNA variable arm binding sites overlap on the C-terminal domain of LeuRS. It was shown that the overhang location and length of the duplexes are important features that LeuRS recognizes. It was hypothesized that LeuRS plays a key role in remodeling specific group I intron ribozymes so that they can productively self-splice.
Issue Date:2016-04-21
Type:Thesis
URI:http://hdl.handle.net/2142/90934
Rights Information:Copyright 2016 Zhongyi Li
Date Available in IDEALS:2016-07-07
Date Deposited:2016-05


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