Residues in the Conserved His Domain of Fruit Fly tRNase Z that Function in Catalysis are Not Involved in Substrate Recognition or Binding

York College of The City University of New York, 94-20 Guy R. Brewer Blvd, Jamaica, NY 11451, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 08/2005; 350(2):189-99. DOI: 10.1016/j.jmb.2005.04.073
Source: PubMed


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.

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Available from: Neela Zareen, Dec 09, 2015
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    • "The 3′ terminal CCA sequence of tRNA is essential for the protein synthesis on ribosome (Green & Noller 1997). As almost all eukaryotic tRNA genes do not encode the 74 CCA 76 sequence, this sequence needs to be added by tRNA nucleotidyltransferase onto the discriminator nucleotide, which is believed to be exposed through removing a 3′ trailer of pre-tRNA by tRNase Z (Deutscher 1990; Nashimoto 1997; Takaku et al. 2003; Vogel et al. 2005; Zareen et al. 2005). However, percentages of the CCA-coding tRNA genes in bacteria and archaea vary with species from 0% to 100%, and the mechanism to remove 3′ trailers and to generate the CCA termini appears to change accordingly (Minagawa et al. 2004). "
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    ABSTRACT: In most organisms, tRNase Z is considered to be essential for 3' processing of tRNA molecules. The Escherichia coli tRNase Z gene, however, appears to be dispensable under normal growth conditions, and its existence remained an enigma. Here we intensively examined various (pre-)tRNAs for good substrates of E. coli tRNase Z in vitro, and found that the enzyme can remove the 3' terminal CCA residues from mature tRNAs regardless of their nucleotide modifications. Furthermore, we discovered that E. coli tRNase Z, when sufficiently expressed in the cell, can shut down growth probably by removing amino acids from aminoacyl-tRNAs. We confirmed in vitro that E. coli tRNase Z exceptionally possesses the activity that cleaves off the 3' terminal residues charging an amino acid from an aminoacyl-tRNA molecule. The current data suggest that tRNase Z might help modulate a cell growth rate by repressing translation under some stressful conditions.
    Full-text · Article · Oct 2008 · Genes to Cells
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    • ", A and C, the residues displayed are those that were subjected to Ala-scanning analysis (14 in the PxKxRN loop and 18 in the Motif I region). Wild-type and variant fruit fly tRNase Z's were constructed, baculovirus-expressed, and affinity-purified (Fig. 3; Zareen et al. 2005; see Materials and Methods). "
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    ABSTRACT: 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.
    Full-text · Article · Jul 2006 · RNA
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    • "Bacteria and archaea genomes contain a tRNase ZS gene only, while eukaryotic genomes encode either only tRNase ZL or both forms. The C-terminal half region of tRNase ZL has high similarity to the whole region of tRNase ZS, and these regions contain a well-conserved histidine motif, which has been shown to be essential for the Mg2+-dependent enzymatic activity by mutational analyses for T.maritima tRNase ZS (10) and Drosophila melanogaster tRNase ZL (13). "
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    ABSTRACT: Thermotoga maritima tRNase Z cleaves pre-tRNAs containing the 74CCA76 sequence precisely after the A76 residue to create the mature 3′ termini. Its crystal structure has revealed a four-layer αβ/βα sandwich fold that is typically found in the metallo-β-lactamase superfamily. The well-conserved six histidine and two aspartate residues together with metal ions are assumed to form the tRNase Z catalytic center. Here, we examined tRNase Z variants containing single amino acid substitutions in the catalytic center for pre-tRNA cleavage. Cleavage by each variant in the presence of Mg2+ was hardly detected, although it is bound to pre-tRNA. Surprisingly, however, Mn2+ ions restored the lost Mg2+-dependent activity with two exceptions of the Asp52Ala and His222Ala substitutions, which abolished the activity almost completely. These results provide a piece of evidence that Asp-52 and His-222 directly contribute the proton transfer for the catalysis.
    Full-text · Article · Feb 2006 · Nucleic Acids Research
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