Structure/function analysis of yeast ribosomal protein L2

Abstract

Ribosomal protein L2 is a core element of the large subunit that is highly conserved among all three kingdoms. L2 contacts almost every domain of the large subunit rRNA and participates in an intersubunit bridge with the small subunit rRNA. It contains a solvent-accessible globular domain that interfaces with the solvent accessible side of the large subunit that is linked through a bridge to an extension domain that approaches the peptidyltransferase center. Here, screening of randomly generated library of yeast RPL2A alleles identified three translationally defective mutants, which could be grouped into two classes. The V48D and L125Q mutants map to the globular domain. They strongly affect ribosomal A-site associated functions, peptidyltransferase activity and subunit joining. H215Y, located at the tip of the extended domain interacts with Helix 93. This mutant specifically affects peptidyl-tRNA binding and peptidyltransferase activity. Both classes affect rRNA structure far away from the protein in the A-site of the peptidyltransferase center. These findings suggest that defective interactions with Helix 55 and with the Helix 65-66 structure may indicate a certain degree of flexibility in L2 in the neck region between the two other domains, and that this might help to coordinate tRNA-ribosome interactions.

Figure 1.

Crown view of the large ribosomal subunit. Ribosomal proteins L2, L3, L4 and L16 (eukaryotic ribosomal protein L10) are indicated. PTC denotes the peptidyltransferase center. CP denotes the central protuberance. The image was generated with PyMol using the yeast cryo-EM-based ribosome mapped onto the H. marismoutui X-ray crystal structure (13).

Figure 2.

The L2 mutants promote numerous phenotypic defects. (A) ‘Killer’ virus phenotypes. The Killer⁺ phenotype is scored by the presence of a halo of growth inhibition around wild-type colony. Lack of the halo around colonies expressing the V48D, L125Q and H215Y L2 mutants indicates the Killer⁻ phenotype. (B) Ten-fold dilutions of indicated cells were spotted onto rich medium and incubated at the indicated temperatures. (C) Yeast cell growth was monitored for 40 h at 30°C with a Synergy HT micro-plate reader and growth curves were generated from four independent readings utilizing the KC4 software package. (D) Drug-sensitivity phenotypes. To monitor changes in sensitivity to paromomycin and anisomycin, 10-fold serial dilutions of each strain were spotted onto rich medium containing these drugs at the indicated concentrations. Sensitivity to sparsomycin was monitored using 6-mm Whatman filter discs saturated with 30 µg of sparsomycin placed onto the center of plates seeded with OD₅₉₅ = 0.2 of yeast cells expressing the indicated L2 mutants.

Figure 3.

The L2 mutants promote specific defects in translational fidelity. Isogenic yeast cells expressing either wild-type or mutant forms of L2 were transformed with the dual luciferase reporter and control plasmids shown in Supplementary Figure S1 and rates of translational recoding were determined. −1 PRF indicates percent of programmed −1 ribosomal frameshifting promoted by the yeast L-A virus frameshift signal. +1 PRF denotes programmed +1 ribosomal frameshifting directed by the frameshift signal of the Ty1 retrotransposable element. Nons-suppr. denotes the percentage of ribosomes able to suppress an in-frame UAA termination codon located between the Renilla and firefly luciferase reporter genes. p values are indicated above samples showing statistically significant changes. Error bars denote SE as previously described (32).

Figure 4.

The V48D and L125Q mutants promote strong 60S biogenesis and subunit-joining defects. Cytoplasmic extracts from isogenic strains were loaded onto 7–47% sucrose gradients, centrifuged in an SW41 rotor at 40 000 r.p.m. for 180 min at 4°C, fractionated and analyzed by continuous monitoring of A₂₅₄ (48). The locations of 40S, 60S, 80S, polysome fractions and half-mers are labeled. The presence of half-mers is indicative of subunit-joining defects.

Figure 5.

The L2 mutants affect tRNA binding and peptidyltransferase activity. (A) Dissociation constants generated by analysis of single-site-binding isotherms of eEF-1A stimulated binding of [¹⁴C]Phe-tRNA to ribosomal A-sites.(B) Dissociation constants generated by analysis of single-site-binding isotherms of Ac-[¹⁴C]Phe-tRNA to ribosomal P-sites. (C) Characterization of peptidyltransferase activities of wild-type and mutant ribosomes generated by analyses of first order time plots of Ac-[¹⁴C]Phe-puromycin formation. Error bars denote mean and standard deviation.

Figure 6.

Structure probing of wild-type and mutant ribosomes. (A) Autoradiograms of reverse transcriptase primer extension reactions spanning sequence in helices 90–73. Sequencing reactions (left sides of panels) are labeled corresponding to the rRNA sense strand. Below each panel, U stands for untreated, D is DMS, C denotes CMCT and K indicates kethoxal. Sources of ribosomes are indicated at top. Protected and deprotected bases are indicated by open and filled arrows, respectively. Chemical modification results in strong stops 1-nt 5′ of the base, and bases with altered chemical protection patterns are identified at left. (B) Localization of bases in the vicinity of the peptidyltransferase center (PTC) of yeast 25S rRNA whose chemical modification patterns were affected by the L2 mutants. (C) Mapping of the L2 mutants and rRNA protection data into the yeast ribosome structure generated by cryo-EM and fitted onto the H. marismortui large subunit crystal structure (13). L2, the PTC, the 3′ end of the peptidyl–tRNA and rRNA helices are labeled. The V48D and L125Q mutants map to the globular domain of L2 where they interact with helices 55 and 65/66, respectively. The H215Y mutant maps to the tip of the extended domain where it inserts into the major groove of helix 93. Nucleotides with altered chemical protection patterns are shown in shades of green. Base numbering follows the S. cerevisiae sequence shown in panel (B).