Investigation of the Roles of the Highly Conserved G-1 Residue of tRNAHis, and Analysis of Unexpected Modification Changes in tRNAHis Variants in Saccharomyces cerevisiae

Abstract

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2009.tRNAs are highly processed molecules that are crucial for protein synthesis in the cell. During tRNA processing, about 13 post-transcriptional modifications are added to each tRNA, many of which are highly conserved across all kingdoms of life. The extra guanine nucleotide at the 5’ end of tRNAHis (G-1) is virtually universally conserved, and tRNAHis is unique since it is the only tRNA to have this G-1 residue. In prokaryotes and some archaea, G-1 is encoded in the genome and retained after 5’ end processing. However, in eukaryotes and other archaea, G-1 is added posttranscriptionally by tRNAHis guanylyltransferase (Thg1), which is highly conserved and essential in Saccharomyces cerevisiae. Although it is known that G-1 is important for tRNAHis aminoacylation, no other function has been ascribed to this almost universally conserved tRNA modification. Thg1 may have other important roles in the cell beyond G-1 addition activity. Thg1 orthologs in yeast and humans are implicated in the cell cycle, particularly in mitosis. Interestingly, Thg1 can also catalyze a 3’ to 5’ (reverse) polymerization reaction in vitro, in which Thg1 adds guanine and cytidine residues to the 5’ end of RNA substrates, in the opposite direction of all other known polymerases. Because of the different possible functions of Thg1, the essential role of Thg1 in the cell is unclear. To study the functions of Thg1 and G-1 of tRNAHis in yeast, nonsense suppression assays were utilized to optimize tRNAHis function in a condition where Thg1 cannot efficiently recognize tRNAHis. Overexpression of both the tRNAHis amber suppressor and histidyl-tRNA synthetase (HTS1) drastically improve nonsense suppression. Surprisingly, the lethality of a thg1-Δ strain can be bypassed by overexpression of both HTS1 and wild-type tRNAHis A73 or tRNAHis C73. Since tRNAHis species from thg1-Δ strains lack G-1, these results demonstrate that the G-1 residue itself is not essential in yeast; however, since thg1-Δ strains are viable under these conditions, the essential function of Thg1 is G-1 addition to tRNAHis. Also surprising is that thg1-Δ strains are healthier with tRNAHis C73 than with tRNAHis A73, despite the fact that eukaryotes have largely conserved A73 on tRNAHis. However, since Thg1 can catalyze reverse polymerization on tRNAHis C73 in vivo, A73 may have been conserved to prevent this activity. Interestingly, thg1-Δ strains are sensitive to paromomycin, an aminoglycoside that induces misreading in the decoding center of the ribosome. Thus, G-1 may be important for mRNA decoding during translation. Further analysis of tRNAHis from thg1-Δ strains demonstrates that 2’-Omethylguanosine (Gm) is reduced in both tRNAHis A73 and tRNAHis C73, and additional 5-methylcytidine (m5C) is present in tRNAHis C73. Upon additional examination, Gm levels are reduced in several conditions where Thg1 activity, and thus tRNAHis function, is compromised: when THG1 is deleted; when the tRNAHis GUG anticodon is mutated to amber; and when A73 is mutated to C73. Furthermore, Gm levels are restored to wild-type levels in the absence of Thg1 when tRNAHis function is improved by mutating residues in the acceptor stem, suggesting that tRNAHis function is important for Gm modification. Analysis of the factors influencing m5C levels reveals that m5C accumulates on tRNAHis C73 variants expressed in either wild-type or thg1-Δ strains, but m5C accumulates on tRNAHis A73 only in conditions where Thg1 activity is depleted. These data are intriguing because they indicate that tRNAHis modifications are sensitive to tRNA function, tRNA sequence, and Thg1 activity. To study other roles of Thg1 in vivo, temperature-sensitive thg1 mutants were generated. These mutants are likely defective in different Thg1 activities since several thg1ts strains display intragenic complementation. These strains will be valuable in the future to dissect the mechanistic steps of both G-1 addition and reverse polymerization