Like all other RNAs, transfer RNA is transcribed as a precursor and must undergo maturation. RNase P removes the 5' end leader. The endonuclease tRNase Z, a member of the (3-lactamase family of metal-dependent hydrolases, can remove the 3' end trailer. CCA can then be added by tRNA nucleotidyltransferase. CCA at the 3' end is a tRNase Z anti-determinant which ensures that mature tRNA proceeds smoothly through aminoacylation. Residues in Motif II, a His cluster (HxHxDH) which is the signature sequence of the (3-lactamase superfamily, are required for tRNase Z catalysis, but not for substrate pre-tRNA binding. Mutagenesis and recent crystal structures of tRNase Z suggest the involvement of residues in homology blocks upstream and downstream of Motif II in metal ion coordination, substrate binding and function of CCA in anti-determination/cleavage site selection. We propose to systematically scan 2 homology blocks of tRNase Z by substitution mutagenesis to establish the mechanism of CCA anti-determination. Additionally, naturally occurring mutations in the pre-tRNA 3' end trailer directly following the tRNase Z cleavage site in human mitochondrial tRNASer(UCN) are associated with maternally transmitted diseases such as non-syndromic deafness. These mutant sequences, like 3'-CCA, could interfere with a productive tRNase Z-substrate interaction. This hypothesis will be investigated by analyzing wild type and mutant substrates. The experimental procedures, including mutagenesis by overlap-extension PCR, baculovirus expression and affinity purification of mutant tRNase Z, Michaelis-Menten analysis of processing kinetics, gel shift determination of Kd for pre-tRNA-tRNase Z complexes, structure probing of pre-tRNAs, photo-crosslinking to identify specific contacts between enzyme and substrate and fluorescence methods to evaluate movement of both enzyme and substrate in the complex, will lead to detailed insight into the regulation of a biomedically significant enzyme reaction. Transfer RNA (tRNA) is central to the process of protein synthesis. Mutations in tRNAs are associated with maternally transmitted mitochondrial diseases and syndromes. Additionally, the gene that encodes 1 of the enzymes in the tRNA maturation pathway has been associated with an elevated risk of prostate cancer, making the proposed research biomedically relevant. ? ? ?
