A flexible loop in yeast ribosomal protein L11 coordinates P-site tRNA binding

Abstract

High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 'P-site loop'. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site.

Figure 1.

Localization of L11 within the ribosome. (A) Image of the yeast ribosome. The large subunit is colored green, and the small subunit is pink. L11 (cyan) is located in the central protuberance of the large subunit where it interacts with 5S rRNA, Helix 84 of 25S rRNA, the T-loop of the peptidyl-tRNA, and the small subunit protein S18 via the B1b and B1c intersubunit bridges. (B) Close-up view of L11 and neighboring structures. Amino acids of L11′s P-site loop targeted for mutation (R51-R61) are colored deep blue. (C) L11 P-site loop amino acids, mutations analyzed in the current study, and their viabilities as the sole form of L11. Y52* represents multiple mutations: Δ, A, R, E, S, I, Q, N, H and F. Ribosomal structures generated in PyMol using yeast cryo-EM (4) with tRNAs from T. thermophilus (5).

Figure 2.

Phenotypic analyses of the viable L11 mutants. (A) 10-fold dilutions of indicated yeast strains were spotted onto SD–Trp media and incubated at temperatures indicated, or (B) on SD–Trp media containing paromomycin, anisomycin or sparsomycin at the indicated concentration and grown at 30°C. (C) Killer virus assays. Wild-type (WT) Killer⁺ cells are identified by a zone of growth inhibition. 51-4A, 54-7A and F57A mutants lack this halo, indicating the Killer⁻ phenotype.

Figure 3.

The L11B mutants promote defects in translational fidelity. Isogenic yeast cells expressing either wild-type or mutant forms of L11B were transformed with dual luciferase reporters and control plasmids and rates of translational recoding were determined. All results are graphed as fold wild type. −1 PRF was measured using the yeast L-A virus frameshift signal. +1 PRF was directed by the frameshift signal derived from the Ty1 retrotransposable element. Nonsense suppression denotes the percentage of ribosomes able to suppress an in-frame UAA termination codon positioned between the Renilla and firefly luciferase reporter genes. Missense suppression rates were evaluated by incorporation of an arginine (AGA) near-cognate amino acid instead of a cognate serine (AGC) at the catalytic codon 218 within the firefly luciferase gene. Error bars denote standard error. P-values are indicated above samples showing statistically significant changes.

Figure 4.

The L11B mutants promote opposing affinities for tRNAs. (A) Binding of tRNA to the P-site. Seventeen picomoles of salt-washed ribosomes were incubated for 40 min at 30°C with 2-fold dilutions of N-acetylated-[¹⁴C]Phe-tRNA and poly(U). 80S-tRNA-poly(U) complexes were bound to nitrocellulose filters and washed with binding buffer. Samples were read by radioactive scintillation counting. Curves were generated using GraphPad Prism 4. (B) P-site tRNA binding K_d values were determined using one site binding with ligand depletions equation. Error bars show standard errors. (C) First-order time plots of multiple turnover peptidylpuromycin reaction. Ribosomes pre-bound with N-acetylated-[¹⁴C]Phe-tRNA and poly(U) were incubated with puromycin. ‘x’ equals percentage of tRNA reacted. (D) Fold wild-type K_obs rates of peptidylpuromycin product formation. Error bars show standard deviations. (E) Binding of tRNA to the A-site. Salt-washed ribosomes were pre-incubated at 30°C with tRNA^Phe to block the P-site, then incubated for 35 min with [¹⁴C]Phe-tRNA plus binding factors and poly(U) as described for P-site binding. (F) K_d values for A-site tRNA binding. Error bars show standard errors.

Figure 5.

L11 mutants promote local and distant changes in rRNA structure. (A) 1M7 SHAPE modification of wild-type and mutant salt washed ribosomes show opposite effects of solvent accessibility on H84 bases C2675-A2679 in mutants 51-4A and 54-7A relative to wild type. DMSO lanes are unmodified controls. Sequencing ladder is shown to the left. (B) PyMol generated image of H84′s protected/deprotected bases (black with gray surface) in mutants 51-4A and 54-7A respectively, for salt-washed empty ribosomes. P-site tRNA is added for reference. Blue spheres mark amino acids changed to alanines in 51-4A, red for 54-7A, purple is mutated in both. (C) Changes in H84 accessibility upon binding of P-site tRNA. Ac-Phe-tRNA^Phe was pre-bound to salt washed ribosomes along with poly(U) and complexes were probed with 1M7 or DMSO controls. In both wild type and 54-7A base C2675 became more deprotected with tRNA bound to the P-site while bases A2676-A2679 show increased protection. 51-4A’s level of protection is unchanged. (D) Differences observed, in both treated and untreated lanes, of natural stops between wild-type and mutant strains in the terminal loop of 25S rRNA Helix 88 (A2779, A2780), and in Expansion Segment 31 (G2531, G2534). (E) Two-dimensional structure of yeast 25S rRNA showing locations of bases showing changes in reactivity. H84 gray highlighted bases protected/deprotected relative to wild-type in empty salt-washed ribosomes in the 51-4A and 54-7A mutants, respectively. Open circled bases indicate sites of decreased innate lability (C2531, G2534) in ES31 for both 51-4A and 54-7A, or increased natural lability (A2779-A2780) in H88 for the 51-4A mutant.

Figure 6.

Model: the P-site loop acts as a sensor of the occupancy status of the P-site. (Left) When the large subunit P-site is unoccupied by tRNA, the L11 P-site loop is able to extend into this space leaving the distal loop of H84 partially deprotected from chemical attack. This conformation is favored by the 54-7A mutant of L11B. (Right panel) Occupation of the P-site by peptidyl-tRNA displaces the L11 P-site loop, causing it to tightly retract from the P-site and interact with H84, resulting in increased protection of the H84 terminal loop from chemical attack. H84 likely moves toward the P-site loop slightly, increasing the exposure of C2675 to the surrounding solvent. This conformation is favored by the L11B 51-4A mutant.