Analysis of the functional modules of the tRNA 3' endonuclease (tRNase Z).
tRNA 3' processing is one of the essential steps during tRNA maturation. The tRNA 3'-processing endonuclease tRNase Z was only recently isolated, and its functional domains have not been identified so far. We performed an extensive mutational study to identify amino acids and regions involved in dimerization, tRNA binding, and catalytic activity. 29 deletion and point variants of the tRNase Z enzyme were generated. According to the results obtained, variants can be sorted into five different classes. The first class still had wild type activity in all three respects. Members of the second and third class still formed dimers and bound tRNAs but had reduced catalytic activity (class two) or no catalytic activity (class three). The fourth class still formed dimers but did not bind the tRNA and did not process precursors. Since this class still formed dimers, it seems that the amino acids mutated in these variants are important for RNA binding. The fifth class did not have any activity anymore. Several conserved amino acids could be mutated without or with little loss of activity.
Späth
Bettina
B
Molekulare Botanik, Universität Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany.
Kirchner
Silvia
S
Vogel
Andreas
A
Schubert
Sylvia
S
Meinlschmidt
Petra
P
Aymanns
Simone
S
Nezzar
Jamel
J
Marchfelder
Anita
A
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology*; D001407:Bacillus / Q000201:enzymology; D002384:Catalysis; D020134:Catalytic Domain; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004252:DNA Mutational Analysis; D019281:Dimerization; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D017353:Gene Deletion; D014644:Genetic Variation; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008670:Metals / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D002614:Chelating Agents; D003432:Cross-Linking Reagents; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D006639:Histidine; D012313:RNA; D012343:RNA, Transfer; D004722:Endoribonucleases; C110250:RNase Z