Residues in two homology blocks on the amino side of the tRNase Z His domain contribute unexpectedly to pre-tRNA 3' end processing

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.

FIGURE 1.

Alignment of selected homology blocks in tRNase Z^L and tRNase Z^S proteins. (A) The PxKxRN loop, (B) the region including Motif I, and (C) the region surrounding Motif II. The top five aligned sequences are tRNase Z^L’s from Drosophila melanogaster, Homo sapiens, Arabidopsis thaliana, Caenorhabditis elegans, and Saccharomyces cerevisiae (accession nos. Q8MKW7, NP_060597.3, AAM51378, O4476, and NP013005.1, respectively). The bottom three sequences are tRNase Z^S’s from B. subtilis, T. maritime, and E. coli (accession nos. P54548, NP_228673, and ZP_00726790, respectively). Designations below the tRNase Z^L panels indicate the consensus, and coloring of residues indicates the extent of homology (green, identical; red, conserved in most cases; purple, similar). Numbering of selected residues above tRNase Z^L panels is for fruit fly tRNase Z^L based on the presumed translation initiation at an internal methionine (r24) as previously described in Zareen et al. 2005. Spacing between the three homology blocks presented is also conserved in the five tRNase Z^L’s. Residue numbers and designations above the bottom panel (indicating the positions of the secondary structure elements within the homology blocks) are based on Bsu tRNase Z^S (de la Sierra Gallay et al. 2005).

FIGURE 2.

Views of tRNase Z taken from the B. subtilis structure (de la Sierra-Gallay et al. 2005; PDB ID 1Y44). Motif II and the other active site residues (Motifs III–V) are buried in the catalytic domain of the A subunit, and the PxKxRN loop appears to cover the entrance to the active site. (A) PxKxRN loop and Motif II. (B) entire Bsu. tRNase Z structure model in the same front view as in A with the external binding domain in the upper left. (C) Motif I region and Motif II; the best view was obtained by rotating ∼180° so that the external binding domain is in the upper right (a back view). The amino acid, α-helix, and β-sheet nomenclature refer to the B. subtilis structure. Residue numbers in parentheses refer to the fruit fly protein.

FIGURE 3.

Baculovirus expression of fruit fly tRNase Z. Wild-type and mutant fruit fly tRNase Zs (indicated with –Z to the right of gel panel) were affinity-purified, electrophoresed on a 10% polyacrylamide SDS gel, stained with Sypro orange (Molecular Probes), and scanned for fluorescence with a Typhoon imager. The variant tRNase Z's presented (K446A, R448A, and Q474A, as designated below panel) were selected based on their effects on processing (see Fig. 3). Approximately 0.5 μg of each tRNase Z sample was loaded. The enzyme concentrations used in kinetic experiments were determined from the protein gel lanes with ImageQuant to obtain k_cat from V_max. Size designations are given for the marker lane to the left of the panel. Fruit fly tRNase Z displays an apparent molecular weight of ∼90 kDa, slightly larger than its predicted molecular weight of 83 kDa.

FIGURE 4.

Kinetics of processing with wild-type and variant tRNase Z. Mutations analyzed are the same as in Figure 2. (A,B) The processing in a kinetic experiment with wild-type and R448A tRNase Z. Reactions were sampled after 5, 10, and 15 min as designated by the brackets below the data panels. The concentrations of tRNase Z (12.5 pM for wild type and 4.5 nM for R448A, designated above the panels) were chosen to produce a similar extent of processing (% product/minute of reaction, equivalent to V/[S]). The substrate concentrations, adjusted by adding unlabeled fruit fly pre-tRNA^His, were 2, 5, 10, 20, and 50 nM, as indicated below the panels. (C) Eadie-Hofstee plots for wild type, K446, R448, and Q474. Concentrations of enzymes used are indicated on the plot. (D) Kinetic parameters from the data presented in A–C. Equations presented for the enzymes (columns labeled –k_M, V_max, with units of × 10⁻⁹ M and × 10⁻¹¹ M/min., respectively) are taken from the Excel plots (C). k_cat is calculated from V_max using the value for [E] determined from the protein gel lane for each mutant (Fig. 1) based on the dilution factor used in the experiment.

FIGURE 5.

Graphical representation of the effects of substitutions in the PxKxRN loop on tRNase Z processing. (A) k_M relative to wild type (from column 3 in Table 1) is presented on a linear scale. The wild-type residues substituted with alanine in the PxKxRN loop are identified on the abscissa. The line and the bar above each value on the bar graph indicate the standard error (from Table 1). (B) The inverse of k_cat relative to wild type (n-fold decrease in k_cat, taken from column 2 in Table 1) is presented on a log scale. (C) The inverse of k_cat/k_M relative to wild type (taken from column 5 in Table 1) is presented on a log scale. Conserved residues (taken from Fig. 1A) that affect one or more of the kinetic parameters are presented with symbols (□,○ for the intermediate effects on k_M and k_cat, respectively; X,@ for the greatest effects). Error bars are not presented in B and C but can be obtained from Table 1.

FIGURE 6.

Graphical representation of the effects of substitutions in the Motif I region. The presentation of k_M, k_cat, and reaction efficiency (k_cat/k_M) relative to wild type in A–C and use of the symbols □,○,■,● is the same as in Figure 5.

FIGURE 7.

Effect of the CCA anti-determinant on efficiency of tRNase Z reaction. T7 runoff templates were designed to produce a pre-tRNA^His substrate with the natural sequence A–AUG or with A–CCA of mature tRNA following the discriminator base (A–). Unlabeled pre-tRNAs were transcribed, gel-purified, treated with shrimp alkaline phosphatase, labeled with γ-³²P-ATP using T4 polynucleotide kinase, and repurified (side designation S for substrate). These labeled pre-tRNAs were used in processing reactions sampled after 5, 10, and 15 min as indicated with vertical ticks below the product bands using enzyme concentrations of 0.0125 and 0.033 nM in the reactions with the –AUG pre-tRNA and with 0.0125, 0.033, 0.083, 0.25, 0.83, and 2.5 nM in reactions with –CCA mature tRNA as indicated below the gel. Comparable processing of –AUG substrate with 0.033 nM tRNase Z and of –CCA tRNA with 2.5 nM tRNase Z suggests that the CCA anti-determinant reduces reaction efficiency ∼80-fold.

FIGURE 8.

A pre-tRNA can be divided into five parts, each of them expected to contribute differently to tRNase Z recognition and catalysis. (A) The T arm; (B) acceptor stem; (C) tRNase Z processing site; (D) three nucleotides following the cleavage site (xxx = CCA of mature tRNA or “not-CCA” of a pre-tRNA); (E) additional sequence of the 3′-end trailer.