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1. The Dynamics of Subunit Rotation in a Eukaryotic Ribosome

   https://doi.org/10.3390/biophysica1020016  

   Freitas, Frederico Campos; Fuchs, Gabriele; de Oliveira, Ronaldo Junio; Whitford, Paul Charles (June 2021, Biophysica)

   null (Ed.)

   Protein synthesis by the ribosome is coordinated by an intricate series of large-scale conformational rearrangements. Structural studies can provide information about long-lived states, however biological kinetics are controlled by the intervening free-energy barriers. While there has been progress describing the energy landscapes of bacterial ribosomes, very little is known about the energetics of large-scale rearrangements in eukaryotic systems. To address this topic, we constructed an all-atom model with simplified energetics and performed simulations of subunit rotation in the yeast ribosome. In these simulations, the small subunit (SSU; ∼1 MDa) undergoes spontaneous and reversible rotation events (∼8∘). By enabling the simulation of this rearrangement under equilibrium conditions, these calculations provide initial insights into the molecular factors that control dynamics in eukaryotic ribosomes. Through this, we are able to identify specific inter-subunit interactions that have a pronounced influence on the rate-limiting free-energy barrier. We also show that, as a result of changes in molecular flexibility, the thermodynamic balance between the rotated and unrotated states is temperature-dependent. This effect may be interpreted in terms of differential molecular flexibility within the rotated and unrotated states. Together, these calculations provide a foundation, upon which the field may begin to dissect the energetics of these complex molecular machines. 

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2. Interactions between terminal ribosomal RNA helices stabilize the Escherichia coli large ribosomal subunit

   https://doi.org/10.1261/rna.079690.123  

   Nissley, Amos J; Kamal, Tammam S; Cate, Jamie H (July 2023, RNA)

   The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro . RNA tags in the Escherichia coli large ribosomal (50S) subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes. 

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3. Shape changes and cooperativity in the folding of the central domain of the 16S ribosomal RNA

   https://doi.org/10.1073/pnas.2020837118  

   Hori, Naoto; Denesyuk, Natalia A.; Thirumalai, D. (March 2021, Proceedings of the National Academy of Sciences)

   Both the small and large subunits of the ribosome, the molecular machine that synthesizes proteins, are complexes of ribosomal RNAs (rRNAs) and a number of proteins. In bacteria, the small subunit has a single 16S rRNA whose folding is the first step in its assembly. The central domain of the 16S rRNA folds independently, driven either by Mg²⁺ions or by interaction with ribosomal proteins. To provide a quantitative description of ion-induced folding of the ∼350-nucleotide rRNA, we carried out extensive coarse-grained molecular simulations spanning Mg²⁺concentration between 0 and 30 mM. The Mg²⁺dependence of the radius of gyration shows that globally the rRNA folds cooperatively. Surprisingly, various structural elements order at different Mg²⁺concentrations, indicative of the heterogeneous assembly even within a single domain of the rRNA. Binding of Mg²⁺ions is highly specific, with successive ion condensation resulting in nucleation of tertiary structures. We also predict the Mg²⁺-dependent protection factors, measurable in hydroxyl radical footprinting experiments, which corroborate the specificity of Mg²⁺-induced folding. The simulations, which agree quantitatively with several experiments on the folding of a three-way junction, show that its folding is preceded by formation of other tertiary contacts in the central junction. Our work provides a starting point in simulating the early events in the assembly of the small subunit of the ribosome. 

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4. Engineered ribosomes with tethered subunits for expanding biological function

   https://doi.org/10.1038/s41467-019-11427-y  

   Carlson, Erik D.; d’Aquino, Anne E.; Kim, Do Soon; Fulk, Emily M.; Hoang, Kim; Szal, Teresa; Mankin, Alexander S.; Jewett, Michael C. (September 2019, Nature Communications)

   Abstract Ribo-T is a ribosome with covalently tethered subunits where core 16S and 23S ribosomal RNAs form a single chimeric molecule. Ribo-T makes possible a functionally orthogonal ribosome–mRNA system in cells. Unfortunately, use of Ribo-T has been limited because of low activity of its original version. Here, to overcome this limitation, we use an evolutionary approach to select new tether designs that are capable of supporting faster cell growth and increased protein expression. Further, we evolve new orthogonal Ribo-T/mRNA pairs that function in parallel with, but independent of, natural ribosomes and mRNAs, increasing the efficiency of orthogonal protein expression. The Ribo-T with optimized designs is able to synthesize a diverse set of proteins, and can also incorporate multiple non-canonical amino acids into synthesized polypeptides. The enhanced Ribo-T designs should be useful for exploring poorly understood functions of the ribosome and engineering ribosomes with altered catalytic properties. 

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5. A steric gate controls P/E hybrid-state formation of tRNA on the ribosome

   https://doi.org/10.1038/s41467-020-19450-0  

   Levi, Mariana; Walak, Kelsey; Wang, Ailun; Mohanty, Udayan; Whitford, Paul C. (December 2020, Nature Communications)

   null (Ed.)

   Abstract The ribosome is a biomolecular machine that undergoes multiple large-scale structural rearrangements during protein elongation. Here, we focus on a conformational rearrangement during translocation, known as P/E hybrid-state formation. Using a model that explicitly represents all non-hydrogen atoms, we simulated more than 120 spontaneous transitions, where the tRNA molecule is displaced between the P and E sites of the large subunit. In addition to predicting a free-energy landscape that is consistent with previous experimental observations, the simulations reveal how a six-residue gate-like region can limit P/E formation, where sub-angstrom structural perturbations lead to an order-of-magnitude change in kinetics. Thus, this precisely defined set of residues represents a novel target that may be used to control functional dynamics in bacterial ribosomes. This theoretical analysis establishes a direct relationship between ribosome structure and large-scale dynamics, and it suggests how next-generation experiments may precisely dissect the energetics of hybrid formation on the ribosome. 

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