Exploration of the conserved A+C wobble pair within the ribosomal peptidyl transferase center using affinity purified mutant ribosomes

Abstract

Protein synthesis in the ribosome's large subunit occurs within an active site comprised exclusively of RNA. Mutational studies of rRNA active site residues could provide valuable insight into the mechanism of peptide bond formation, but many of these mutations cause a dominant lethal phenotype, which prevents production of the homogeneous mutant ribosomes needed for analysis. We report a general method to affinity purify in vivo assembled 50S ribosomal subunits containing lethal active site mutations via a U1A protein-binding tag inserted onto the 23S rRNA. The expected pH-dependent formation of the A2450+C2063 wobble pair has made it a potential candidate for the pH-dependent conformational change that occurs within the ribosomal active site. Using this approach, the active site A2450+C2063 pair was mutated to the isosteric, but pH-independent, G2450*U2063 wobble pair, and 50S subunits containing the mutations were affinity purified. The G*U mutation caused the adjacent A2451 to become hyper-reactive to dimethylsulfate (DMS) modification in a pH-independent manner. Furthermore, the G*U mutation decreased both the rate of peptide bond formation and the affinity of the post-translocation complex for puromycin. The reaction rate (k(pep)) was reduced approximately 200-fold for both puromycin and the natural aminoacyl-tRNA A-site substrate. The mutations also substantially altered the pH dependence of the reaction. Mutation of this base pair has significant deleterious effects upon peptidyl transferase activity, but because G*U mutation disrupts several tertiary contacts with the wobble pair, the assignment of A2450 as the active site residue with the neutral pK(a) important for the peptidyl transferase reaction cannot be fully supported or excluded based upon these data.

Figure 1

(A) Schematic representation of an A+C (upper panel) and a G•U wobble pair (lower panel). In this study the A2450+C2063 pair is mutated to a G•U pair. (B) Diagram of the U3X-tag inserted into the 23S rRNA for affinity purification. The three U1A-binding sites are delineated in gray.

Figure 2

Affinity purification of U3X-tagged 50S ribosomes. (A) Selective retention of U3X-tagged 50S ribosomes by His₆U1A protein. Ribosomes or ribosomes containing a U3X-tag were affinity purified by incubation with Ni-NTA and His₆U1A protein. Aliquots of the batch purification process were analyzed by SDS–PAGE, as indicated, and stained with Coomassie Blue. (B) Analysis of ribosome purity by primer extension. Lane 1, no template. Lane 2, untagged rRNA control. Lane 3, homogeneous U3X-tagged rRNA control. Lanes 4–6, affinity purified 23S rRNA from U3X-tagged wt or G•U wobble mutant (GU) ribosomes. Lanes 5 and 6 are from two independent large-scale purifications of G•U ribosomes. Lane 7, G•U mutant 23S rRNA from ribosomes in the translocation complex.

Figure 3

Kinetic analysis of G•U mutant ribosomes. (A) Rate of tripeptide formation in the reaction between fMetPhe-tRNA^Phe and Pmn. The extent of reaction is plotted as a function of time for wt 50S ribosomes prepared using standard protocols from pop2136 (closed circles) or MRE600 (open circles) cells, U3X-tagged wt 50S ribosomes (closed squares) and U3X-tagged G•U mutant ribosomes (open squares) prepared by affinity purification. The reaction was normalized to the final amplitudes of the extent of tripeptide formation. (B) Concentration dependence of Pmn reaction (37°C, pH 7.2). Further increase in concentration was not feasible due to limited solubility of Pmn. (C) pH dependence of the G2450•U2063 affinity purified mutant ribosomes at 10 mM Pmn (37°C). The reaction rate is plotted versus the pH in the range 6.2–8.3. (D) Rate of dipeptide formation in the reaction between fMet-tRNA^fMet and Phe-tRNA^Phe. The extent of reaction is plotted as a function of time for wt 50S ribosomes (closed circles) and U3X-tagged G•U mutant ribosomes (open circles) prepared by affinity purification. The reaction is normalized to the final amplitudes of the extent of dipeptide formation.

Figure 4

DMS modification of G2450•U2063 mutant ribosomes as a function of pH. Ribosomes were incubated at pH 6.0 or 8.5 in the absence (−) or presence (+) of DMS. The 23S rRNA was purified and used as a template for primer extension of a radiolabeled primer. The resulting products were separated by denaturing gel electrophoresis. RT stops at nucleotides C2452 and A2451 are indicated.

Figure 5

Structure of conserved residues within the 23S rRNA active site from the H.marismortui 50S subunit crystal structure (2). The A2450+C2063 wobble pair (E.coli numbering) is shown in orange, A2451 is shown in blue, G2061 and G2447 are shown in green and C2501 is in yellow. The N1 of A2451 that reacts with DMS is shown as an enlarged sphere. The hydrogen bonds between A2450+C2063 that are likely to be disrupted by mutation to a G•U pair are shown in red. This includes interactions within the A2450+C2063-C2501 triple, as well as the G2061-A2451-G2447 triple stacked above it.