Diverse Biological Functions For 3'-5' Nucleotide Addition Reactions: tRNA Repair to tRNAHis Identit

Transfer RNAs (tRNAs), are crucial for the process of gene expression due to their adaptor function in translation. Emerging evidence suggests that regulatory functions of tRNAs in gene expression might extend well beyond their role in polypeptide synthesis. tRNAs are highly processed prior to attaining mature status. These steps include 5'-and 3'-end processing, splicing and modification, all of which are required either for optimal activity and/stability of mature tRNAs in vivo. As a result, processed tRNAs undergo surveillance that ensure high quality of mature tRNAs. Such surveillance pathways, collectively referred to as quality control pathways, actively degrade misprocessed tRNAs before they enter the translational pool. In this thesis, we have identified two mechanisms that complement these well-studied quality control pathways by instead positively reinforcing the optimal function of tRNAs and therefore are broadly classified as quality assurance mechanisms.;The quality assurance pathways that are the focus of this work are intimately linked to the tRNAHis guanylyltransferase (Thg1) family, which comprises a unique class of enzymes that catalyze 3'-5' nucleotide addition, opposite to all other DNA and RNA polymerases. The Thg1 enzymes are presumed essential in all eukaryotes due to their strict requirement for eukaryotic tRNAHis maturation because of their function in G-1 addition to tRNAHis. G-1 is a highly conserved tRNA His identity element across all three domains of life. Yet, here we reveal several instances of cytosolic and mitochondrial tRNAHis biogenesis mechanisms that are not dependent on G-1, and hence Thg1, for establishment of tRNAHis identity. Alternatively, we also report a Thg1-independent mechanism for G-1 acquisition in C. elegans, which is the first instance of a bacteria-like mechanism for incorporating G-1 in eukaryotes. Taken together, these observations indicate a more substantial diversity in the mechanisms for G-1 acquisition and tRNAHis identity in eukaryotes than was previously thought based on the widespread occurrence of Thg1 enzymes in initial analyses.;This documented diversity in tRNAHis identity pathways underscores the possibility that G-1 addition does not appear to have been the ancient function of Thg1 enzymes. Members of the Thg1 family are found in all three domains of life, yet functions of Thg1-like proteins (TLPs) found in Bacteria and Archaea were unknown. Using in vitro biochemical assays we demonstrated that the activities of TLPs from the bacterium Bacillus thuringiensis (BtTLP) and archaeon, Methanosarcina acetivorans (MaTLP) are more suited for a function in tRNA 5'-end repair. Moreover, we have also demonstrated that the activity of BtTLP is not restricted to tRNA substrates but extends to other RNAs, including 5S rRNA, suggesting the possibility of even broader role for 3'-5' polymerases in RNA quality assurance.;Finally, we have undertaken an investigation into the suitability of using the 3'-5' addition activity of Thg1 enzymes to develop a novel RNA 5'-end labeling technique. This technique allows site specific addition of standard and non-standard nucleotide analogs to RNA 5'-ends, the utilities for which are many in the field of RNA biochemistry and structural biology.