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A fluorescence-based approach to elucidate the subunit arrangement of the essential tRNA deaminase from Trypanosoma brucei

Winner, Katherine M.
http://rave.ohiolink.edu/etdc/view?acc_num=wuhonors1617803573189193

Abstract Details

2019, Bachelor of Arts, Wittenberg University, Biochemistry/Molecular Biology.
Trypanosoma brucei, a parasitic protozoan, is the etiologic agent of African Sleeping Sickness (Trypanosomiasis), a disease that threatens the lives of thousands in sub-Saharan Africa, with a 100% mortality rate in untreated cases. Transfer RNA (tRNA) is an adaptor molecule which acts as the intermediary between nucleotide and amino acid sequences, linking the information encoded in the genome to protein synthesis. Naturally occurring nucleotide modifications of tRNA are critical for translation; and therefore, essential for the viability of the organism. RNA editing involves the chemical modification of one nucleotide to another; in T. brucei, some tRNAs undergo essential editing events from adenosine to inosine. The heterodimeric enzymes, called adeonsine deaminases acting on tRNA (ADAT2/3), catalyzes the deamination of the first position of the anticodon in tRNA. Inosine in the wobble base position allows a single tRNA to decode multiple codons, implicating ADAT2/3 as a potential anti-parasitic therapeutic target. This enzyme consists of two structurally unique subunits, ADAT2 and ADAT3, both of which bind Zn2+. Given its essentiality in parasitic viability, it is important to understand the enzyme’s structure and function. Since the structure of ADAT2/3 has been difficult to elucidate, a Tripartite Split-Green Fluorescent Protein (GFP) Association Assay was used to determine the correct orientation of each subunit in vitro. This assay uses GFP split into three fragments, which fluoresce when they are all in close proximity to one another. By strategically tagging the termini of the enzyme with the two small linker fragments, and incubating with the larger reporter, the correct orientation of the ADAT2/3 heterodimer can be determined on the basis of fluorescence of a reassembled GFP. To validate the results of the assay, a general cross linking experiment, followed by mass spectrometry, should also be conducted. Ultimately, elucidation of ADAT2/3 structure is important to understand this modification pathway at a molecular level, potentially leading to the development of anti-trypanosomal drugs that will help combat this medically important parasite. Co-author: Alfonzo, Juan D.
Margaret Goodman (Advisor)
Michelle McWhorter (Committee Member)
Daniel Marous (Committee Member)
24 p.
Biochemistry; Microbiology; Molecular Biology; Parasitology
Trypanosoma brucei; African Sleeping Sickness; Trypanosomiasis, African; Transfer RNA; tRNA editing; ADAT2; ADAT3; Tripartite Split-Green Fluorescent Protein Association Assay; GFP; fluorescence

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Citations

  • Winner, K. M. (2019). A fluorescence-based approach to elucidate the subunit arrangement of the essential tRNA deaminase from Trypanosoma brucei [Undergraduate thesis, Wittenberg University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=wuhonors1617803573189193

    APA Style (7th edition)

  • Winner, Katherine. A fluorescence-based approach to elucidate the subunit arrangement of the essential tRNA deaminase from Trypanosoma brucei. 2019. Wittenberg University, Undergraduate thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=wuhonors1617803573189193.

    MLA Style (8th edition)

  • Winner, Katherine. "A fluorescence-based approach to elucidate the subunit arrangement of the essential tRNA deaminase from Trypanosoma brucei." Undergraduate thesis, Wittenberg University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=wuhonors1617803573189193

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Wittenberg University Honors Theses and OhioLINK.