Transfer RNA (tRNA) is central to protein synthesis. Like other RNAs, tRNA is transcribed as a precursor and must undergo maturation. RNase P removes the 5'leader. The endonuclease tRNase Z removes the 3'trailer so that CCA can be added by tRNA nucleotidyltransferase. tRNase Z reaction is thus a critical step in pre-tRNA maturation. A flexible arm (FA) is extruded from the body of tRNase Z remote from the active site. The globular FA hand principally binds to the elbow (D/T loops) of tRNA, far from the scissile bond. Naturally occurring mutations in human mitochondrial tRNAs are associated with maternally transmitted diseases and syndromes, principally myopathies. [Aim 1] Does the distribution of pathogenesis-related mitochondrial tRNA mutations correlate with tRNA structure changes and effects on tRNase Z processing? Effects of pathogenesis-related T loop substitutions will be investigated. [Aim 2] Does protease susceptibility report on flexibility of the tRNase Z FA? The mass spectroscopic analysis will shed light on a novel mechanism of substrate recognition. Enter the text here that is the new specific aims information for your application. One page is recommended. tRNAs are transcribed as precursors and processed by removal of a 5'leader and a 3'trailer. RNase P endonucleolytically removes the 5'leader. tRNase Z, the pre-tRNA 3'processing endonuclease, cleaves the 3'trailer leaving an OH on the discriminator, the unpaired nucleotide at the 3'side of the acceptor stem, prepared for CCA addition. tRNase Z is a member of the ?- lactamase family of metal-dependent hydrolases. Ability to specifically recognize tRNA is uniquely conferred upon tRNase Z by the flexible arm (FA), a globular hand extruded from and connected to the body of the enzyme by a structured stalk, which contacts nucleotides in the T- loop of substrate tRNA (Li et al., 2005, 2006).
Aim 1. Does the distribution of pathogenesis-related mitochondrial tRNA mutations correlate with tRNA structure changes and effects on tRNase Z processing? Analysis of effects on tRNase Z reaction of pathogenesis-related mutations in the T-loops of mitochondrial tRNAs will be combined with tRNA structure probing. 11 of the 22 human mitochondrially encoded tRNAs harbor 13 pathogenesis-related T-loop substitutions (see MITOMAP: A Human Mitochondrial Genome Database;www.mitomap.org, 2011). To investigate patterns of effects on structure, processing and the resulting pathologies, we will construct these wild type and mutant mitochondrial tRNAs and characterize effects of the substitutions on their structure and tRNase Z processing kinetics.
Aim 2. Does protease susceptibility report on flexibility of the tRNase Z FA? A deletion study demonstrated function of the FA in pre-tRNA substrate recognition (Schilling et al., 2005). The FA contributes two orders of magnitude toward substrate binding and specific FA residues and regions are important for binding (Levinger et al., 2009). To investigate flexibility of the FA, we will use protease susceptibility and mass spectrometry to compare wild type tRNase Z with several important variants, both free in solution and complexed with pre-tRNA.
Mutations in mitochondrial tRNAs are associated with maternally transmitted diseases and syndromes. Moreover, the gene that encodes tRNase Z has been associated with an elevated risk of prostate cancer. tRNA is central to translation and removal of the pre-tRNA 3'trailer by tRNase Z is central to tRNA maturation. Experimental procedures including mutagenesis by overlap-extension PCR, baculovirus expression and affinity purification of variant tRNase Z, in vitro transcription and labeling to prepare pre-tRNA substrates, analysis of binding and processing kinetics, structure probing of pre-tRNAs, and spectroscopic analysis will lead to detailed insight into function of a biomedically significant enzyme.