Residues in two homology blocks on the amino side of the tRNase Z His domain contribute unexpectedly to pre-tRNA 3' end processing.
tRNase Z, which can endonucleolytically remove pre-tRNA 3'-end trailers, possesses the signature His domain (HxHxDH; Motif II) of the beta-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 3' end approximately 80 times less efficiently than a pre-tRNA possessing natural sequence of the 3'-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.
Zareen
Neela
N
York College of The City University of New York, Jamaica, 11451, USA.
Hopkinson
Angela
A
Levinger
Louis
L
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D001618:beta-Lactamases / Q000378:metabolism
D029721:Drosophila Proteins; D012322:RNA Precursors; D006639:Histidine; D012343:RNA, Transfer; D001120:Arginine; D004722:Endoribonucleases; C502293:tRNase Z, Drosophila; D001618:beta-Lactamases; D000409:Alanine; D005998:Glycine