RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase

Abstract

In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.

FIGURE 1.

RNA molecules used in this study. tRNA^His is from E. coli. The sequence of duplex^His is based on that of the minihelix^His. However, in the duplex the following was done: (i) U⁶⁵ (as numbered in native tRNA^His) was changed to a C to improve the stability of the duplex; (ii) the G⁵²–C⁶² base pair was changed to C⁵²–G⁶² to prevent hybridization of two duplex molecules through their free 3′-ends, and (iii) the G¹–C⁷² base pair was changed to C¹–G⁷² to prevent formation of a triple helix in NMR conditions (see text). These changes are shown in red. At the base of the loop of the tetraloop^His, a G-C base pair (in cyan) was introduced to reinforce the tetraloop stability. As shown at the bottom of the figure, two tetraloop^His molecules (in black and in green) may associate to form a triple helix. Resulting Hoogsteen G-C⁺ base pairs in the triple helix are indicated by stars.

FIGURE 2.

HSQC spectra of His-tagged Pth-H20A in the presence of various concentrations of duplex^His. The ¹H-¹⁵N HSQC spectrum obtained in the presence of 130 μm Pth alone (in red) is superimposed to those obtained after addition of duplex^His at an RNA/protein ratio of 0.4 (in blue) or 0.8 (in yellow). Experiments were performed at 17 °C, in a 50 mm sodium acetate buffer (pH 6.0) containing 200 mm NaCl. Shown by arrows are the H^N–N peaks of several residues sensitive to the presence of the RNA.

FIGURE 3.

Chemical shift and intensity variations of H^N–N peaks of Pth-H20A upon addition of duplex^His. For each residue (1–193) of the protein, the figure shows the chemical shift variation (Δδ = ((ΔδH^N)² + (ΔδN/10)²)^1/2) and the IPI upon addition of duplex^His at an RNA/protein ratio of 0.4. Residues for which the Δδ value is higher than 0.02 ppm are in blue. Residues for which the IPI value is higher than 0.8 are in yellow. Residues for which both the Δδ value is higher than 0.02 ppm and the IPI value is higher than 0.8 are in green. A, amide protons of the protein backbone. In the histogram, a Δδ value of 0 was given to the proline residues and to the superimposed peaks for which the detection of the chemical shift variation was impossible. B, amide protons of the asparagines and glutamine side chains.

FIGURE 4.

Pth-H20A residues affected by RNA binding. A, on the molecular surface of Pth (PDB accession number 2PTH, chain A) on which hydrogen atoms have been added, residues for which the Δδ value is higher than 0.02 ppm are in blue; residues for which the IPI value is higher than 0.8 are in yellow, and residues for which both the Δδ value is higher than 0.02 ppm and the IPI value is higher than 0.8 are in green. The protein is displayed on two faces differing by 180°. The locations of several surface residues whose resonances are affected by RNA binding are shown by arrows. B, schematic representations of the protein with the same orientations and colors as in A, showing α-helices and β-strands. The figure was generated with PyMol (DeLano Scientific, San Carlos, CA).

FIGURE 5.

Region of the ¹⁵N-HSQC spectra corresponding to the H^ϵ protons of the Pth arginine side chains. A, effect of duplex^His addition. Spectra were recorded in the presence of 130 μm Pth in the absence of RNA (in red) or in the presence of RNA at an RNA/protein ratio of 0.4 (blue) or 0.8 (yellow). B, effect of the R133A mutation. The spectrum of the H20A mutant is in red and that of the H20A/R133A double mutant is in green. The peaks of two H^ϵ protons, resonating at lower frequencies, are not visible in the figure. These peaks are not significantly modified either by the addition of duplex^His or by the R133A mutation.

FIGURE 6.

Schematic view of the complex between duplex^His and Pth-H20A, based on the best-scored solution of a CNS energy minimization procedure. α-Helices and β-strands are shown with protein residues colored as in Fig. 4B. Side chains of Phe-66, Lys-105, Arg-133, and Lys-182 residues are in red. RNA backbone is in orange.