The 9-A solution: how mRNA pseudoknots promote efficient programmed -1 ribosomal frameshifting

Abstract

There is something special about mRNA pseudoknots that allows them to elicit efficient levels of programmed -1 ribosomal frameshifting. Here, we present a synthesis of recent crystallographic, molecular, biochemical, and genetic studies to explain this property. Movement of 9 A by the anticodon loop of the aminoacyl-tRNA at the accommodation step normally pulls the downstream mRNA a similar distance along with it. We suggest that the downstream mRNA pseudoknot provides resistance to this movement by becoming wedged into the entrance of the ribosomal mRNA tunnel. These two opposing forces result in the creation of a local region of tension in the mRNA between the A-site codon and the mRNA pseudoknot. This can be relieved by one of two mechanisms; unwinding the pseudoknot, allowing the downstream region to move forward, or by slippage of the proximal region of the mRNA backwards by one base. The observed result of the latter mechanism is a net shift of reading frame by one base in the 5' direction, that is, a -1 ribosomal frameshift.

FIGURE 1.

Overview of programmed –1 ribosomal frameshifting. (Top) An mRNA pseudoknot induces elongating ribosomes to pause with their A- and P-site tRNAs positioned over the heptameric X XXY YYZ ‘‘slippery site’’ (red arrow). The incoming frame is indicated by spaces. (Middle) While paused at the slippery site, if the ribosome shifts by 1 base in the 5′ direction, the non-wobble bases of both the A- and P-site tRNAs can re-pair with the new −1 frame codons. (Bottom) The mRNA pseudoknot is denatured (arrow), and elongation continues in new reading frame.

FIGURE 2.

The mRNA pseudoknot from Beet Western Yellows Virus (Egli et al. 2002). Stem 1 residues are depicted in green, loops and bulging residues in yellow, stem 2 residues are shown in red, and the 3′ base is colored lavender. We attached a 6-nt linker RNA (orange) and A- and P-site codons (blue) modeled to fit into the downstream mRNA tunnel of the Thermus thermophilis 30S ribosomal subunit (Yusupova et al. 2001). Figure constructed using the Swiss-Pdb Viewer.

FIGURE 3.

Docking of the mRNA pseudoknot crystal structure from the Beet Western Yellows Virus –1 PRF signal into the mRNA tunnel of the Thermus thermophilis 30S subunit (Ogle-etal-2001; Yusupova-etal-2001). The rRNA is shown as a gray ribbon; ribosomal proteins are white except for S3, S4, and S5, which are pink; the BWYV mRNA pseudoknot is green; the 6-nt linker in the mRNA tunnel is orange; the A- and P-site codons are shown as blue. Figures were constructed using the Swiss-Pdb Viewer. (A) View from the intersubunit side of the 30S subunit. S3 is to the left and behind the mRNA pseudoknot, S4 is at the top right, and S5 is partially obscured by rRNA at the middle right. (B) 50-Å cutaway section with S3 and S5 to the left, and S4 to the right. (C) View from shoulder side of 30S subunit. S3 is on the top, S4 is on the bottom, and S5 is behind the pseudoknot. (D) 50-Å cutaway section of view shown in panel C.

FIGURE 4.

The 9-Å solution. (Top) The 0-frame A- and P-site codons of a programmed −1 ribosomal frameshift signal are base-paired to cognate peptidyl- and aa-tRNAs occupying the P/P and A/T hybrid states respectively. (Middle) Upon accommodation, the anticodon loop of the aa-tRNA moves 9 Å in the 5′ direction, pulling the 3′ mRNA sequence along with it. The mRNA pseudoknot is too large to enter the downstream tunnel, with the consequence that the linker region between the A-site codon and the mRNA pseudoknot is stretched, creating a localized region of tension in the linker mRNA. (Bottom) Decoupling of the A- and P-site codon:anticodon interactions from the 0-frame, and re-pairing in the −1 frame repositions the mRNA so as to relieve the tension.