A conformational change in the ribosomal peptidyl transferase center upon active/inactive transition

Abstract

The ribosome is a dynamic particle that undergoes many structural changes during translation. We show through chemical probing with dimethyl sulfate (DMS) that conformational changes occur at several nucleotides in the peptidyl transferase center upon alterations in pH, temperature, and monovalent ion concentration, consistent with observations made by Elson and coworkers over 30 years ago. Moreover, we have found that the pH-dependent DMS reactivity of A2451 in the center of the 23S rRNA peptidyl transferase region, ascribed to a perturbed pKa of this base, occurs only in inactive 50S and 70S ribosomes. The degree of DMS reactivity of this base in the inactive ribosomes depends on both the identity and amount of monovalent ion present. Furthermore, G2447, a residue proposed to be critical for the hypothesized pKa perturbation, is not essential for the conditional DMS reactivity at A2451. Given that the pH-dependent change in DMS reactivity at A2451 occurs only in inactive ribosomes, and that this DMS reactivity can increase with increasing salt (independently of pH), we conclude that this observation cannot be used as supporting evidence for a recently proposed model of acid/base catalyzed ribosomal transpeptidation.

Figure 1

The peptidyl transferase center. (A) Secondary structure of the peptidyl transferase center (PTC), with bases of interest in this study indicated. All numbering is for E. coli. (B) Schematic of the catalytic center taken from the H. marismortui 50S crystal structure, with the relative position of the tetrahedral intermediate analogue soaked into the crystal indicated (16).

Figure 2

pH-dependent changes in DMS reactivity at A2451 in 70S ribosomes under different conditions. Primer extension analysis of 23S rRNAs isolated from unmodified (DMS −) and DMS-modified (DMS +) ribosomes. A, C, and U are dideoxy sequencing lanes. (A) Influence of DMS incubation conditions: 70S ribosomes were modified with DMS either for 10 min at 37°C or for 1 h at 0°C. The asterisk denotes A2469, the modification of which is strongly enhanced at higher temperatures. (B) Influence of the activation of 70S ribosomes by preincubation for 15 min at 37°C (lanes 1–4) or 15 min at 0°C (lanes 5–8), before DMS modification (1 h at 0°C).

Figure 3

Influence of changing monovalent ion concentration and temperature on the 50S subunit's pH-dependent DMS reactivity at A2451 and ability to perform peptidyl transfer. Unm, unmodified ribosomes. (A) Heat activation of 50S subunits in K⁺ (lanes 6–8) or NH (lanes 12–14) results in loss of pH-dependent DMS reactivity at A2451. (B) Heat incubation in the absence of monovalent cation (lanes 6–8) or in the presence of Na⁺ (lanes 12–14) does not result in loss of pH-dependent DMS reactivity at A2451. (C) Increasing concentrations of K⁺ (lanes 3–5) or NH (lanes 6–8) at constant pH (6.5) at 0°C results in increasing DMS reactivity of A2451. (D) 50S subunits shown to exhibit pH-dependent DMS reactivity at A2451 cannot perform peptidyl transferase as assayed by the fragment reaction. 50S preincubation conditions are as shown.

Figure 4

Mutation of G2447 to A has no effect on pH-dependent DMS reactivity of A2451. Unm, unmodified ribosomes. Lanes 3–5: WT ribosomes. Lanes 6–8: G2447A ribosomes. Sequencing lanes are given for the G2447A mutant.

Figure 5

Changes in DMS reactivity at additional nucleotides in the peptidyl transferase center on active/inactive transition. pH-dependent DMS reactivity of A2572 and U2584 changes on going from active (lanes 2–4) to inactive (lanes 6–8) ribosomes.