Figure 1.
Location of L11 in 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 B1 b and B1 c intersubunit bridges. (B) Close-up view of L11 and neighboring structures. Amino acids of L11′s intersubunit region targeted for mutation are colored orange, red, and pink, corresponding to the colored amino acid shown. Ribosomal structures generated in PyMol using yeast cryo-EM [5] fitted with tRNAs from T. thermophilus [6].
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. Inability to maintain the Killer+ phenotype indicates altered translational fidelity.
Figure 3.
The L11 B1 b/c bridge mutants affect 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 measures the percentage of ribosomes suppressing an in-frame UAA termination codon positioned between the Renilla and firefly luciferase reporter genes. Missense suppression rates evaluated near-cognate utilization of a tRNAArg tRNA at an AGC serine codon at position 218 within the firefly luciferase gene. Error bars denote standard error. Asterisks above samples indicate statistically significant changes as determined by t-test.
Figure 4.
The L11B mutants affect binding affinities for tRNAs.
(A) Binding of tRNA to the A-site. Ribosomal P-sites were blocked with tRNAPhe at 30°C, then incubated for 35 minutes with [14C]Phe-tRNA plus elongation factors and poly(U). 80 S-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) A-site tRNA binding KDs were determined using one site binding with ligand depletion equation. Error bars depict standard deviation. (C) Binding of tRNA to the P-site. Ribosomes were incubated for 40 minutes at 30°C with dilutions of N-acetylated-[14C]Phe-tRNA and poly(U) and processed as described for A-site binding. (D) KDs for P-site tRNA binding. Error bars depict standard deviation.
Figure 5.
L11 mutants promote local and distant changes in rRNA structure.
(A)1 M7 SHAPE modification of 25S rRNA for wild-type and mutant puromycin treated salt washed ribosomes. DMSO lanes are unmodified controls. Sequencing ladders are shown to the left of each panel. (B) SHAPE modification for 18 S rRNA for same mutant ribosomes. All mutants were probed multiple times, and representative images are presented. Only regions with consistent effects are displayed here.
Figure 6.
Location of altered rRNA bases.
(A) Location of altered bases in two dimensional structure of 25S rRNA, and (B) in the 18 S rRNA. (C) Position of structural changes in three dimensional structure of the ribosome in two separate views. Light blue surface is large subunit rRNA, deeper blue surface are proteins, 5S rRNA is purple, light gray surface is 18 S rRNA, darker gray are proteins. The E, P, and A site tRNAs are orange, black, and deep blue respectively. L11 is in cyan, S18 in yellow, H84 plus extension is lime-yellow. Deprotected bases are shown as red spheres, while bases with increased levels of protection are green spheres.
Figure 7.
Models describing the B1 b/c bridge and its role in transmitting information between the ribosomal subunits.
(A) Proposed “wiring diagram.” Overlaid arrows depict communication pathway connecting the decoding center (DC) in the SSU to the PTC in the LSU. Coloration is identical to that used in Figure 6C. (B) Cartoon depicting how charge-charge interactions mediate transition of the B1 b/c bridge through an orderly series of allosteric ratcheting states.