tRNAs are transcribed as precursors with a 5 end leader and a 3 end trailer. The 5 end leader is processed by RNase P, and in most organisms in all three kingdoms, transfer ribonuclease (tRNase) Z can endonucleolytically remove the 3 end trailer. Long ( L ) and short ( S ) forms of the tRNase Z gene are present in the human genome. tRNase Z L processes a nuclear-encoded pre-tRNA ϳ1600-fold more efficiently than tRNase Z S and is predicted to have a strong mitochondrial transport signal. tRNase Z L could, thus, process both nuclear-and mitochondrially encoded pre-tRNAs. More than 150 pathogenesis-associated mutations have been found in the mitochondrial genome, most of them in the 22 mitochondrially encoded tRNAs. All the mutations investigated in human mitochondrial tRNA Ser(UCN) affect processing efficiency, and some affect the cleavage site and secondary structure. These changes could affect tRNase Z processing of mutant pre-tRNAs, perhaps contributing to mitochondrial disease.Mitochondria, the intracellular organelles responsible for most of a eukaryotic cell energy production, possess their own maternally inherited genome. The circular 16,568-base pair human mitochondrial genome (1) is bidirectionally transcribed into long polycistronic heavy (H) 4 -and light (L)-strand precursor RNAs. Two rRNAs and 11 mRNAs encoding 13 polypeptides (all of them subunits of complexes in the respiratory transport chain) are punctuated by 22 tRNAs (1 tRNA for each of 18 amino acids and 2 each for leucine and serine with different anticodons) with very little intergenic RNA (2, 3). Several thousand additional polypeptides required for mitochondrial metabolism are nuclear-encoded, cytoplasmically translated and imported.Precursor tRNA transcripts have a 5Ј end leader and a 3Ј end trailer. Eukaryotic nuclear-encoded tRNAs are transcribed from RNA polymerase III promoters with short 5Ј end leaders and 3Ј end trailers ending with a U 3 tail. The characteristic length and sequence of mitochondrial pre-tRNA leaders and trailers, on the other hand, depend on the setting of individual tRNAs among the neighboring functional RNA units (other tRNAs, mRNAs, rRNAs) on the long polycistronic H-and L-strand transcripts (4).The 5Ј end leader is removed from pre-tRNAs by RNase P in a largely conserved, well characterized reaction (5). On the other hand, there are different paths to a mature tRNA 3Ј end (6). In the tRNAs of some prokaryotes, CCA is transcriptionally encoded, and the 3Ј end trailer is removed by the sequential activity of an endonuclease (RNase E) and exonucleases (7,8). In contrast, in most organisms in all three kingdoms and in extranuclear organelles, CCA is not transcriptionally encoded and can be added to the discriminator (the unpaired nucleotide after the 3Ј end of the acceptor stem, which is retained in mature tRNA) after precise endonucleolytic removal of the 3Ј end trailer by tRNase Z (9 -14).Intriguingly, CCA is a tRNase Z anti-determinant that prevents mature tRNA from recycling through tRNase Z, thus ensuring that ...
tRNase Z, which can endonucleolytically remove pre-tRNA 39-end trailers, possesses the signature His domain (HxHxDH; Motif II) of the b-lactamase family of metal-dependent hydrolases. Motif II combines with Motifs III-V on its carboxy side to coordinate two divalent metal ions, constituting the catalytic core. The PxKxRN loop and Motif I on the amino side of Motif II have been suggested to modulate tRNase Z activity, including the anti-determinant effect of CCA in mature tRNA. Ala walks through these two homology blocks reveal residues in which the substitutions unexpectedly reduce catalytic efficiency. While substitutions in Motif II can drastically affect k cat without affecting k M , five-to 15-fold increases in k M are observed with substitutions in several conserved residues in the PxKxRN loop and Motif I. These increases in k M suggest a model for substrate binding. Expressed tRNase Z processes mature tRNA with CCA at the 39 end ;80 times less efficiently than a pre-tRNA possessing natural sequence of the 39-end trailer, due to reduced k cat with no effect on k M , showing the CCA anti-determinant to be a characteristic of this enzyme.
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