返回Google圖書搜尋

Catalytic and Biological Implications of the Eukaryotic and Prokaryotic Thg1 Enzyme Family

出版	Ohio State University, 2019
URL	http://books.google.com.hk/books?id=isZ1zQEACAAJ&hl=&source=gbs_api

註釋In all, the goal of this thesis is to further elucidate the catalytic mechanism and biological functions of the tRNAHis guanylyltransferase enzyme family. The tRNAHis guanylyltransferase (Thg1) family performs non-canonical 3' to 5' nucleotide addition, which is in the opposite direction of canonical 5' to 3' polymerases. The Thg1 family of enzymes consists of two members, Thg1 and Thg-Like-Proteins (TLP) and is represented in organisms from all domains of life. Though both Thg1 enzymes and TLPs catalyze 3' to 5' addition, Thg1 enzymes perform both non-Watson Crick (non-WC) addition and Watson-Crick (WC) addition. However, TLPs only catalyze strictly templated WC addition. Both Thg1 enzyme family members perform 3' to 5' addition by activating the substrate with ATP, followed by the incorporation of the incoming nucleotide. Interestingly, eukaryotes have harnessed the 3' to 5' non-WC addition activity of Thg1 enzymes to post-transcriptionally add a single guanine to the -1 position of tRNAHis as part of its maturation process. This seemingly modest addition of one nucleotide to tRNAHis ensures translational fidelity by providing a critical identity element for histidyl aminoacyl tRNA synthetase (HisRS). Like HisRS, Thg1 utilizes the anticodon to specifically recognize tRNAHis. Among the residues responsible for this interaction are highly conserved H152 and K187, which have also been shown to be important for catalysis but their exact role has been difficult to determine because substitution of either residue completely abolishes catalytic activity. Using conservative substitutions and single-turnover kinetic assays, we demonstrate that H152 and K187 are responsible for activating tRNAHis for non-WC addition. In addition to its function in tRNAHis maturation, human Thg1 (hThg1) is involved in mitochondria biogenesis. Recently, Thg1's function as a tRNAHis maturation enzymes and its role in the mitochondria has converged in newly identified disease-associated point mutations in hThg1. This thesis presents data on how the biological functions of hThg1 may contribute to genetic neurological disorders. Research regarding the function of Thg1 and TLPs has been largely performed in eukaryotic models due to their genetic tractability. The function of Thg1 enzymes in tRNAHis maturation cannot be assumed because there are examples of organisms that either do not require G-1 or genomically-encode the extra guanylylate. However, some of these organisms encode a TLP enzyme and can theoretically catalyze templated G-1 addition if G-1 is required for histdylation by HisRS. Strikingly, studies in Dictyostelium discoideum have revealed that the 3' to 5' nucleotide addition activity of TLPs can be utilized for multiple different cellular functions. Our studies of TLP activity and biological function in Myxococcus xanthus provides an example of an organism that seemingly requires G-1 but does not use its encoded TLP enzyme for tRNAHis maturation. Instead, the work presented in this thesis supports a role for Myxococcus xanthus TLP in acting on other RNAs that are important for cellular differentiation.