Residues in the conserved His domain of fruit fly tRNase Z that function in catalysis are not involved in substrate recognition or binding.
Transfer RNAs are transcribed as precursors with extensions at both the 5' and 3' ends. RNase P removes endonucleolytically the 5' end leader. tRNase Z can remove endonucleolytically the 3' end trailer as a necessary step in tRNA maturation. CCA is not transcriptionally encoded in the tRNAs of eukaryotes, archaebacteria and some bacteria and must be added by a CCA-adding enzyme after removal of the 3' end trailer. tRNase Z is a member of the beta-lactamase family of metal-dependent hydrolases, the signature sequence of which, the conserved histidine cluster (HxHxDH), is essential for activity. Starting with baculovirus-expressed fruit fly tRNase Z, we completed an 18 residue Ala scan of the His cluster to analyze the functional landscape of this critical region. Residues in and around the His cluster fall into three categories based on effects of the substitutions on processing efficiency: substitutions in eight residues have little effect, five substitutions reduce efficiency moderately (approximately 5-50-fold), while substitutions in five conserved residues, one serine, three histidine and one aspartate, severely reduce efficiency (approximately 500-5000-fold). Wild-type and mutant dissociation constants (Kd values), determined using gel shifts, displayed no substantial differences, and were of the same order as kM (2-20 nM). Lower processing efficiencies arising from substitutions in the His domain are almost entirely due to reduced kcat values; conserved, functionally important residues within the His cluster of tRNase Z are thus involved in catalysis, and substrate recognition and binding functions must reside elsewhere in the protein.
Zareen
Neela
N
York College of The City University of New York, 94-20 Guy R. Brewer Blvd, Jamaica, NY 11451, USA.
Yan
Hua
H
Hopkinson
Angela
A
Levinger
Louis
L
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D029721:Drosophila Proteins; D006639:Histidine; D004722:Endoribonucleases; C502293:tRNase Z, Drosophila