Crystal structure of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshifting pseudoknot

Figures

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  FIGURE 1.. Structure of the SARS-CoV-2 frameshifting pseudoknot. (A) Secondary structure. Arrows denote chain connectivity; Leontis and Westhof (2001) symbols, non-Watson–Crick base pairs. Numbering corresponds to the SARS-CoV-2 reference genome (NC_045512.2), except for residues 39–46 (outlined letters) introduced to facilitate crystallization. (B) Cartoon representation of the 1.3 Å-resolution structure of the pseudoknot, colored as in A. Nucleotides altered for crystallization are gray. Pink spheres denote chain connectivity in lieu of U13534, which is disordered. For alternate conformers, bases are in pastel shades. Red, lime, light purple, and purple spheres represent water molecules, Mg2+, Na+, and K+, respectively. Cobalt hexammine is colored blue.

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  FIGURE 2.. Four base triples produce uninterrupted base stacking between P1 and P2. (A) Network of hydrogen-bonding interactions (black dashes) of the four base triples spanning the P1–P2 junction. Only water molecules bridging nucleobases are shown. Alternate conformers of G13538 and G13539 are light blue. (B) Axial view of the G13486•C13492•G13538 triple. Blue mesh shows a portion of the 2|Fo|–|Fc| simulated annealing-omit map contoured at 1σ. Hydrogen-bonding distances (Å) are indicated. (C) Axial view of the U13485•G13493•A13537 triple. The ribose of U13494 (whose base is extruded into solvent) extends the hydrogen-bonding network. (D) Axial view of the G13484•C13495•C13536 triple. The phosphate of U13494 extends the hydrogen-bonding network. (E) Axial view of the A13483•U13496•A13535 triple.

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  FIGURE 3.. Ribose and ion interactions stabilize pseudoknot. (A) Interactions between the 2′-OH groups of G13493 and U13494 with the A13537 nucleobase are shown, along with the ribose zipper hydrogen bonding between 2′-OH groups of C13495 and C13536. (B) Interactions between a cobalt(III) hexammine with the O6 and N7 of G13493 and G13484. (C) Interactions among a hydrated Mg2+, K+, and four tiers of base pairs within the major groove. Distances (Å) are shown for the oxygen ligand shared between the Mg2+ and K+ in a µ-oxo arrangement. Hydrogen bonds between the hydrated Mg2+ and base pairs are colored black, K+-ligand bonds are represented by dotted red lines, and the µ-oxo bond involving K+ is represented by a thick dotted line. Yellow mesh shows a portion of the anomalous difference Fourier synthesis calculated with 1.771 Å X-ray data (Table 1), contoured at 3 σ.

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  FIGURE 4.. Model for pseudoknot plasticity during translation. Prior to encountering the ribosome, the frameshifting pseudoknot adopts multiple conformations. The pseudoknot structure determined by cryo-EM (Zhang et al. 2021) contains an additional helix (red) from nts 13,459–73, which includes the upstream slippery site and is absent from the coaxially stacked pseudoknot structure presented herein. As the ribosome proceeds past the slippery site, the fully stacked conformation may be favored.

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  Christopher P. Jones.