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. [Aim 3] Might pre-tRNA 3'trailers enter a regulatory network following tRNase Z reaction? We will investigate binding of the pre-tRNA 3'trailer products of tRNase Z cleavage to La and members of the Ago family in early steps of the hypothesized pathway.
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.
| Wilson, Christopher; Ramai, Daryl; Serjanov, Dmitri et al. (2013) Tethered domains and flexible regions in tRNase Z(L), the long form of tRNase Z. PLoS One 8:e66942 |
