A molecular clamp ensures allosteric coordination of peptidyltransfer and ligand binding to the ribosomal A-site

Abstract

Although the ribosome is mainly comprised of rRNA and many of its critical functions occur through RNA-RNA interactions, distinct domains of ribosomal proteins also participate in switching the ribosome between different conformational/functional states. Prior studies demonstrated that two extended domains of ribosomal protein L3 form an allosteric switch between the pre- and post-translocational states. Missing was an explanation for how the movements of these domains are communicated among the ribosome's functional centers. Here, a third domain of L3 called the basic thumb, that protrudes roughly perpendicular from the W-finger and is nestled in the center of a cagelike structure formed by elements from three separate domains of the large subunit rRNA is investigated. Mutagenesis of basically charged amino acids of the basic thumb to alanines followed by detailed analyses suggests that it acts as a molecular clamp, playing a role in allosterically communicating the ribosome's tRNA occupancy status to the elongation factor binding region and the peptidyltransferase center, facilitating coordination of their functions through the elongation cycle. The observation that these mutations affected translational fidelity, virus propagation and cell growth demonstrates how small structural changes at the atomic scale can propagate outward to broadly impact the biology of cell.

Figure 1.

The L3 basic thumb. (A) Crown view of the yeast 60S subunit from (36). L3 is indicated in green. The aa-tRNA accommodation corridor is framed by Helix 89, and the complex structure formed by Helices 90–92. Elongation factors bind to the GTPase Associated Center (GAC) and the Sarcin/Ricin Loop (SRL) at the tip of Helix 95. The peptidyltransferase center (PTC) is in the center of the large subunit. (B) The 3D view of isolated L3, heat map colored from the N-terminus (blue) to the C-terminus (red). The N-terminal extension and the basic thumb and tryptophan (W) finger of the central extension are indicated. (C) The 3D view of interactions between amino acid residues of the L3 basic thumb investigated in this study. It is surrounded by a cagelike structure formed by 25S rRNA Helices 61–64, H73 and H90. Amino acids mutated in this study are labeled.

Figure 2.

Effects of L3 basic thumb mutants on cell growth, virus propagation and programmed –1 ribosomal frameshifting. (A) Ten-fold dilution spot assays (10⁴ → 10⁰ CFU) of cell growth at 30°C. (B) Killer assay. Cells were replica plated onto a lawn of diploid Killer⁻ indicator cells and grown at 20°C for 3 days. Zone of growth inhibition indicates presence of the Killer virus. (C) Dual luciferase assays were used to measure percent programmed –1 ribosomal frameshifting from an L-A virus derived –1 PRF signal (20;21). Error bars denote standard errors.

Figure 3.

Biochemical characterization of ribosomes expressing L3 basic thumb mutants. (A) Single site isotherms of eEF1A stimulated binding of [¹⁴C]Phe-tRNA^Phe to A-sites of poly(U) primed ribosomes pre-loaded with tRNA^Phe in their P-sites. (B) Dissociation constants calculated from data shown in (A). (C) Single site isotherms of Ac-[¹⁴C]Phe-tRNA^Phe to P-sites of poly(U) primed ribosomes. (D) Dissociation constants calculated from data shown in (C). (E) eEF2-binding isotherms for wild-type and mutant ribosomes. (F) Dissociation constants calculated from data shown in (E). (G) Ac-[¹⁴C]Phe-puromycin formation is plotted as percent of bound donor reacted. (H) Rates of peptidylpuromycin formation (K_obs) were calculated from data shown in (G).

Figure 4.

rRNA structure probing of wild-type and L3 basic thumb mutant ribosomes. (A) Poly(U) primed salt-washed ribosomes were either loaded with Ac-Phe-tRNA^Phe (P-site occupied) or tRNA^Phe+ Phe-tRNA^Phe (A + P-site occupied). Ribosomes were unmodified (control) or treated with 1M7 as indicated. Reverse transcriptase primer extension reactions spanned sequences from the 3′ half of Helix 73 through Helix 96 (left panel), or from Helix 91 through the 3′ half of Helix 73 (right panel). Sequencing reactions (left sides of panels) are labeled corresponding to the rRNA sense strand. Nucleotides whose 2′OH riboses were protected from 1M7 modification in mutant relative to wild-type ribosomes of are indicated by white arrowheads, and those deprotected relative to wild-type are indicated by black arrowheads. Bases marked in gray (A2926 and A2971) were deprotected when the A-site is unoccupied relative to when it contains aa-tRNA. (B) rRNA protection patterns of the L3 basic thumb mutants mapped onto the 2D diagram of 25S rRNA. Arrowheads indicate relatively protected and deprotected bases as above. Colored boxes indicate bases that interact with specified L3 basic thumb amino acid side chains. A2926 and A2971 are circled in gray, and C2925, which is the first gate in the aa-tRNA accommodation corridor, is circled in purple. The three bases participating the Type II A-minor motif that stabilizes the PTC are boxed and indicated. (C) Data from panels A and B mapped onto the 3D structure of the yeast ribosome. Indicated bases colored black correspond to bases deprotected in the mutants, while those colored gray are hyperprotected. Bases participating in the Type II A-minor motif (A^m) are colored purple. Helical structures and the PTC are color coded as indicated. Note that the loop formed between H61–H64 was removed from this figure because it obscures the L3 basic thumb.