Optimization of Ribosome Structure and Function by rRNA Base Modification

BACKGROUND: Translating mRNA sequences into functional proteins is a fundamental process necessary for the viability of organisms throughout all kingdoms of life. The ribosome carries out this process with a delicate balance between speed and accuracy. This work investigates how ribosome structure and function are affected by rRNA base modification. The prevailing view is that rRNA base modifications serve to fine tune ribosome structure and function. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, yeast strains deficient in rRNA modifications in the ribosomal peptidyltransferase center were monitored for changes in and translational fidelity. These studies revealed allele-specific sensitivity to translational inhibitors, changes in reading frame maintenance, nonsense suppression and aa-tRNA selection. Ribosomes isolated from two mutants with the most pronounced phenotypic changes had increased affinities for aa-tRNA, and surprisingly, increased rates of peptidyltransfer as monitored by the puromycin assay. rRNA chemical analyses of one of these mutants identified structural changes in five specific bases associated with the ribosomal A-site. CONCLUSIONS/SIGNIFICANCE: Together, the data suggest that modification of these bases fine tune the structure of the A-site region of the large subunit so as to assure correct positioning of critical rRNA bases involved in aa-tRNA accommodation into the PTC, of the eEF-1A•aa-tRNA•GTP ternary complex with the GTPase associated center, and of the aa-tRNA in the A-site. These findings represent a direct demonstration in support of the prevailing hypothesis that rRNA modifications serve to optimize rRNA structure for production of accurate and efficient ribosomes

Sensitivity of rRNA base modification mutants to Translational Inhibitors.

<p>Mutant and isogenic wild-type yeast strains were spotted as ten fold dilutions from 10⁵ to 10¹ CFU onto YPAD media containing 20 µg/ml anisomycin or sparsomycin. Cells were incubated for 3 days at 30°C, and growth was monitored as compared to growth on plates in the absence of drug. Each strain and drug was assayed at least twice.</p

25S rRNA in the peptiptidyl transferase center of yeast.

<p>(A) Secondary structure of yeast 25S rRNA in the PTC. snoRNAs targeted for this study are indicated along with the residues they modify. <i>Ψ</i> – pseudouridylated residue; Nm – 2′-<i>O</i>-ribose methylated residue. Helices are numbered in black. (B) Three dimensional representation of the <i>E. coli</i> PTC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000174#pone.0000174-Schuwirth1" target="_blank">[34]</a>. Modified residues are labeled by the colors indicated in panel A. <i>Left</i>: view into the PTC from the top of the LSU, <i>righ</i>t: 90° rotation of <i>Left</i>. Helices and tRNAs are labeled.</p

Ribosome biochemistry.

<p>Mutant strain <i>snr46</i><i>Δ</i> and the isogenic wild-type are shown in the top row, and mutant strain <i>spb1DA/snr52</i><i>Δ</i> and the isogenic wild-type are show in the bottom row. Error bars represent standard error for all graphs. A. [¹⁴C]Phe-tRNA binding to the A-site of the ribosome. One site binding curves of bound tRNA as analyzed using GraphPad Prism software. Data are reported as a percentage of the total tRNA bound. B. Ac-[¹⁴C]Phe-tRNA binding to the P-site of the ribosome. One site binding curves of bound tRNA as analyzed using GraphPad Prism software. Data are reported as a percentage of the total tRNA bound. C. Peptidyltransfer. Timecourse assays of peptidyltransferase activities as measured by the puromycin reaction.</p

Many of the rRNA base modification mutants have M₁ virus propagation defects.

<p>Yeast rRNA modification mutants were tested for their ability to maintain the L-A and M₁ viruses. (A) Mutant and isogenic wild-type yeast strains were spotted onto YPAD plates, and allowed to grow at 30°C, and then replica plated to a seeded lawn of 5X47 indicator cells. Plates were incubated at room temperature for 3–5 days until a zone of inhibition was clearly visible for wild-type cells. (B) Total RNAs were extracted from mutant and isogenic wild-type yeast strains and digested with RNase A under high salt conditions. The resulting double-stranded RNA was separated on a 1% agarose gel and visualized with ethidium bromide. L-A and M₁ dsRNAs are indicated. The image was inverted for clarity.</p