The 9-Å 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 Å 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.Keywords: Virus; ribosome; translation; genetic code; recoding; structure/function After a generation spent in the shadows, the ribosome is enjoying a renaissance. Recent breakthroughs in X-ray crystallography and cryoelectron microscopy have given us atomic-level views of this complex molecular machine Wimberly et al. 2000;Harms et al. 2001;Spahn et al. 2001;) that are bringing into focus the relationship between ribosome structure and function (Gabashvili et al. 1999;Agrawal et al. 2000;Carter et al. 2000;Frank and Agrawal 2000;Mueller et al. 2000;Nissen et al. 2000;Schluenzen et al. 2000;Beckmann et al. 2001;Nissen et al. 2001; Pioletti et al. 2001;Polacek et al. 2001;Thompson et al. 2001;Yusupova et al. 2001;Noller et al. 2002;Schmeing et al. 2002;Simonson and Lake 2002). One of the major requirements of the ribosome is to maintain translational reading frame, and an increasing number of cis-acting mRNA signals that alter this have been used to probe this essential function of the translational machinery. These translational "recoding" events (Gesteland and Atkins 1996) can take many forms, for example, "slips" of one or more bases, "hops" spanning as many as 50 nucleotides, and "shunts" around large mRNA secondary structures (for review, see Jacks 1990;Brierley 1995;Farabaugh 1996;Giedroc et al. 2000). Programmed −1 ribosomal frameshifting (−1 PRF) is the most widely used translational recoding mechanism of RNA viruses. The −1 PRF signal can be broken down into three discrete parts: the "slippery site", a linker region, and a downstream region of secondary mRNA structure, typically an mRNA pseudoknot. Mutagenesis studies from many different laboratories have demonstrated that the primary sequence of the slippery site and its placement in relation to the incoming translational reading frame is critical: It must be X XXY YYZ, where X must be a stretch of three identical nucleotides, Y is either AAA or UUU, and Z is A, C, or U. Although less is known about the linker region, ...

Mechanical Unfolding of the Beet Western Yellow Virus −1 Frameshift Signal

“…Even though there has been extensive structural and functional characterization of frameshifting pseudoknots (15,16,(20)(21)(22), the mechanism by which pseudoknots regulate -1 PRF remains largely unknown. The ability of a pseudoknot to stimulate frameshifting is defined as its frameshifting efficiency, which is the percentage of the time a frameshift occurs at a site known to produce frameshifting products.…”

“…At these sites, initial codon-anticodon pairing in the zero frame is followed by dissociation and re-pairing of the tRNA anticodons (peptidyl tRNA and aminoacyl tRNA) to mRNA in the -1 reading frame at an overlapping codon (15). The slippery sequence is highly conserved among -1 PRF events, with the general form X XXY YYN where XXX must be three identical nucleotides, Y is either A or U, and Z is A, C, or U (22,42,43).…”

“…In the 9 Å model, movement of 9 Å by the anticodon loop of the aminoacyl-tRNA at the accommodation step is thought to create tension in the mRNA and subsequent slippage of the mRNA in the 5' direction (22). It is known that significant movement of the aa-tRNA occurs during accommodation from the A/T to the A/A state (48).…”

“…The 9 Å model suggests that the downstream mRNA pseudoknot provides resistance to this movement by becoming wedged into the entrance of the ribosomal mRNA tunnel (22). 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.…”

“…(Bottom) The A-and P-site codonanticodon interactions are disrupted from the zero frame and repair in the -1 frame. Image adapted from (22).…”