Extensive Ribosome and RF2 Rearrangements during Translation Termination

Svidritskiy, Egor; Demo, Gabriel; Loveland, A.B.; Xu, Chen; Коростелев, А.A.

doi:10.1101/600445

Cited by 7 publications

(11 citation statements)

References 100 publications

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“…Interestingly, cryo-EM models of the ribosome in complex with release factors (RF) have revealed a tRNA conformation (Structure III 52 , 53 ) that is similar to our identified I1 ensemble (Supplementary Fig. 11 ).…”

Section: Resultssupporting

confidence: 62%

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

et al. 2020

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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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Section: Resultssupporting

confidence: 62%

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

et al. 2020

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“…5E). Similar tRNA states were observed in several termination complexes where they are thought to represent an intermediate to the P/E hybrid-state after deacylation by a release factor (Graf et al, 2018;Svidritskiy et al, 2019) (Supp. Fig.…”

Section: Cryo-em Analysis Reveals Hiv Fss Hairpin Binding To the A Sitementioning

confidence: 54%

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Bao

Loerch

Ling

et al. 2020

Preprint

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Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

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“…For most complexes, grid screening and data collection were performed on the Titan Krios within one microscope session. Data acquisition is performed via multi-hole technique for R1.2/1.3 or smaller grids, or multi-hole and multi-shot approach for grids R2/2 or larger [17].…”

Section: Methodsmentioning

confidence: 99%

“…Although, the method is mostly successful in molecules with rather large molecular weight, cryo-EM has steadily demonstrated success in the sub-MDa range, eventually crossing the 100 kDa threshold [11,12]. Many structures or structure ensembles, unsuccessfully sought after with X-ray crystallography, were solved by cryo-EM [13][14][15][16][17]. In biologics research, a broad range of complexes, such as antigens bound to Fabs or multiple VHH, are typically close to or heavier than 100 kDa and, thus, are suitable targets for cryo-EM [18].…”

Section: Introductionmentioning

confidence: 99%

Development of an integrated structural biology platform specialized for sub-100 kDa protein complexes to support biologics discovery and rational engineering

Iozzo

Qiu

et al. 2021

Preprint

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Developing a biologic medicine requires successful decision making at each step of selection, optimization, and/or combination of the right candidates at early research stages. Knowing the structural information and binding pattern between drug target and discovery candidates greatly increases the possibility of success. With the cryo-EM resolution revolution and rapid development of computational software, we have evaluated and integrated different tools in structural biology and the computation field and established a highly cost-effective platform, which allows us to obtain fast and accurate structural information for biologics projects with a close to 100% success rate and as fast as weeks turn-around time. Here we report four case studies selected from over 40 different protein structures and share how we integrate cryo-EM structure determination, computational structure modeling, and molecular dynamics simulation. With proper decision making and strategic planning, the platform allows us to obtain quality results within days to weeks, including sub-100 kDa complexes which are usually considered as a challenge due to their small size. Our utilization of this differential approach and use of multiple software packages, allows to manage priorities and resources to achieve goals quickly and efficiently. We demonstrate how to effectively overcome particle orientation bias by altering complex composition. In several of our examples, we use glycan density to facilitate interpretation of low-resolution 3D reconstruction and epitope mapping. Protein information plays an important role in our cryo-EM projects, especially in cases where we see significant challenges in obtaining high-resolution 3D maps.

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Extensive Ribosome and RF2 Rearrangements during Translation Termination

Cited by 7 publications

References 100 publications

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

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

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Development of an integrated structural biology platform specialized for sub-100 kDa protein complexes to support biologics discovery and rational engineering

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