Biochemical and genetic studies of mitochondrial protein synthesis in Saccharomyces cerevisiae : characterization of the AEP3 and TRM5 gene products

• Main Author: Lee, Changkeun, 1971-
• Format: Others
• Language: English
• Published: 2012
• Subjects: Saccharomyces cerevisiae > Genetics; Proteins > Synthesis; Mitochondria; Transfer RNA
• Online Access: http://hdl.handle.net/2152/17928

Protein synthesis in archaebacteria and the cytoplasm of eukaryotes is initiated using the initiator methionyl-tRNA (Met-tRNA[subscript i][superscript Met]). In contrast, formylated methionyltRNA (fMet-tRNA[subscript i][superscript Met][subscript f]) is found in eubacteria, and in chloroplasts and mitochondria of eukaryotes, and this formylated initiator tRNA was widely believed to be required for initiation of protein synthesis in those systems. However, the fact that initiation of protein synthesis in yeast mitochondria can occur with unformylated initiator tRNA has changed our perspective about the initiation of mitochondrial protein synthesis. This dissertation is composed of two parts. Part I describes an investigation of the yeast AEP3 gene which was isolated by a genetic screening system in Saccharomyces cerevisiae. The main goal of this part was to discover new accessory factor(s) that might be involved in initiation of protein syntheis of yeast mitochondria when there is no formylation of initiator tRNA and determine how they support the initiation process in Saccharomyces cerevisiae. The synthetic petite genetic screen identified the AEP3 gene. Protein-protein binding assays as well as protein-initiator tRNA binding assays indicate that Aep3p is associated with the initiation process in yeast mitochondrial protein synthesis. This discovery is important because it suggests the possible mechanism by which initiation of protein synthesis in yeast mitochondria occur under conditions where there is no formylation of initiator tRNA. Part II describes a study of the TRM5 gene encoding a tRNA methyltransferase in S. cerevisiae. The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) previously known to methylate guanosine at position 37 (m¹G37) in certain cytoplasmic tRNAs in S. cerevisiae. The main goal of this part was to investigate whether Trm5p is also responsible for m¹G37 modification of mitochondrial tRNAs. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a [Delta]trm5 mutant strain, as well as with a synthetic mitochondrial tRNA[superscript Met][subscript f] and tRNA[superscript Phe]. High pressure liquid chromatography analysis showed the methylated product to be m¹G. Analysis of subcellular fractionation and immunoblotting revealed that the enzyme was localized to both cytoplasm and mitochondria. Our data including the analysis of N-terminal truncation mutants suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis. === text