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Review
. 2009 Feb;139(2):193-208.
doi: 10.1016/j.virusres.2008.06.008. Epub 2008 Jul 25.

Frameshifting RNA Pseudoknots: Structure and Mechanism

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Free PMC article
Review

Frameshifting RNA Pseudoknots: Structure and Mechanism

David P Giedroc et al. Virus Res. .
Free PMC article

Abstract

Programmed ribosomal frameshifting (PRF) is one of the multiple translational recoding processes that fundamentally alters triplet decoding of the messenger RNA by the elongating ribosome. The ability of the ribosome to change translational reading frames in the -1 direction (-1 PRF) is employed by many positive strand RNA viruses, including economically important plant viruses and many human pathogens, such as retroviruses, e.g., HIV-1, and coronaviruses, e.g., the causative agent of severe acute respiratory syndrome (SARS), in order to properly express their genomes. -1 PRF is programmed by a bipartite signal embedded in the mRNA and includes a heptanucleotide "slip site" over which the paused ribosome "backs up" by one nucleotide, and a downstream stimulatory element, either an RNA pseudoknot or a very stable RNA stem-loop. These two elements are separated by six to eight nucleotides, a distance that places the 5' edge of the downstream stimulatory element in direct contact with the mRNA entry channel of the 30S ribosomal subunit. The precise mechanism by which the downstream RNA stimulates -1 PRF by the translocating ribosome remains unclear. This review summarizes the recent structural and biophysical studies of RNA pseudoknots and places this work in the context of our evolving mechanistic understanding of translation elongation. Support for the hypothesis that the downstream stimulatory element provides a kinetic barrier to the ribosome-mediated unfolding is discussed.

Figures

Fig. 1
Fig. 1
Folding topology and solution structural model of the phage T2 gene 32 autoregulatory pseudoknot (PDB 2TPK) in which the two pseudoknot stems S1 and S2 are coaxially stacked on one another (Du et al., 1996; Holland et al., 1999). The small arrow indicates the position of pseudoknot loop L3, when present, while the large vertical arrow indicates the direction of approach of the translocating ribosome.
Fig. 2
Fig. 2
Organization of HIV-1, SARS-CoV and PEMV-1 genomic RNAs highlighting the frameshift sites (fs) in each case. A) Production of viral fusion proteins, e.g., Gag-Pol in HIV-1, from a single translation initiation event (indicated by the pink ribosome) via a −1 PRF (fs) event. B) Schematic rendering of a bipartite frameshift signal.
Fig. 3
Fig. 3
Ribbon representation of the path of the messenger RNA bound to the 30S ribosomal subunit and the three transfer RNAs (PDB 1JGO) (Yusupova et al., 2001). A) The A-site, P-site and E-site tRNAs are indicated (gold), the mRNA is purple, and S3, S4 and S5 proteins are indicated. B) Ribbon representation of the structure of the mRNA entry channel indicating a candidate docking site for RNA elements that stimulate −1 PRF.
Fig. 4
Fig. 4
Close-up view of the decoding center of the small ribosomal subunit taken from an atomic resolution structure (2.8 Å) of the a pre-translocation complex of the 70S bacterial ribosome with bound mRNA and aminoacyl tRNAPhe in the A-site, deacylated initiator tRNAfMet in the P-site and noncognate tRNA in the E-site (Selmer et al., 2006) (PDB 2J02). A) Aminoacyl A-site and deacylated P-site tRNA anticodon-mRNA codon interactions are shown. A Mg2+ ion is also shown which may help induce a kink if the mRNA and therefore maintain reading frame just prior to translocation. B) Close-up of the P- and E-site tRNA codon regions within the decoding center, highlighting the displacement of the P-site tRNA codon toward the A-site. See text for details. Adapted from (Selmer et al., 2006).
Fig. 5
Fig. 5
A schematized view of a single translocation cycle of translation that shows three possible points in the cycle that −1 frameshifting could potentially be stimulated by a downstream pseudoknot. In one scenario, −1 PRF is proposed to occur from the hybrid A/P–P/E state which is stabilized by EF-G•GTP (eEF2•GTP) binding; this binding in turn, weakens the interaction of the deacylated P-site tRNA with the mRNA inducing frameshifting before 30S (40S in eukaryotes) translocation to the classical P/P and E/E states accompanied by hydrolysis and EF-G•GDP (eEF2•GDP) release (Ermolenko et al., 2007; Spiegel et al., 2007). Alternatively, −1 PRF occurs during the 30S (40S) translocation step itself (Namy et al., 2006). Both models are distinguished from a previous model that invoked simultaneous slippage (SS) of two tRNAs in the 5′ direction after accommodation and before peptidyl transfer (Jacks et al., 1988; Plant et al., 2003). See text for additional details.
Fig. 6
Fig. 6
Cryo-electron microscopy (16 Å resolution) of mammalian 80S ribosomes paused at the IBV frameshift signal (Namy et al., 2006). A) Overall structure of the complex with the large (60S) and small (40S) ribosomal subunits indicated as are the P-site tRNA (green), eukaryotic elongation factor-2 (eEF2; red) and the pseudoknot (PK, purple). (B) Close-up of the 40S subunit focusing on the mRNA entry channel and the electron density for the P-site tRNA in the pseudoknot complex vs. that bound to a non-frameshift stimulating stem-loop RNA. Note the clear differences in the position of the P-site tRNA (see text for details). C) A cartoon model of a mechanical model for −1 PRF, in which slippage of the P-site tRNA occurs during translocation mediated by eEF2-GTP hydrolysis. Reproduced with permission from (Namy et al., 2006).
Fig. 7
Fig. 7
Secondary structural representations of structurally or functionally characterized frameshifting elements. Upper panel, three distinct structural classes of canonical two-stem containing hairpin (H)-type pseudoknots, with the luteoviral P1-P2 helical junction region boxed. Lower panel, frameshifting elements that do not conform to the standard H-type pseudoknot paradigm.
Fig. 8
Fig. 8
Stereo views of non-luteoviral P1-P2 frameshifting signals of known three-dimensional structure. A) MMTV gag-pro pseudoknot vpk (1RNK) (Shen and Tinoco, 1995); B) SRV–1 gag-pro pseudoknot (1E95) (Michiels et al., 2001); C) HIV-1 gag-pol stem loop (1Z2J) (Staple and Butcher, 2005b); D) SIV gag-pol stem-loop (2JTP) (Marcheschi et al., 2007). Close-up views of the helical junction regions for the MMTV and SRV-1 gag-pro pseudoknots and the well-ordered hairpin loops for HIV-1 and SIV gag-pol signals are also shown (right). For the pseudoknots, S1 is shaded yellow, S2 is blue, L1 is red, L2 is green and L3 is purple (see also Figs. 9–10). Note that these structures are not drawn to scale.
Fig. 9
Fig. 9
Stereo views of the structures of four related luteoviral P1-P2 pseudoknots. A) BWYV (PDB 1L2X solved to 1.25 Å resolution) (Egli et al., 2002); PLRV (PDB 2A43 solved to 1.34 Å resolution) (Pallan et al., 2005); C) PEMV-1 (PDB 2RP1) (this work); D) ScYLV (PDB 1YG4) (Cornish et al., 2005). Close-ups of the helical junction regions of all four RNAs are shown to the right, with the L2-S1 minor groove base triple highlighted at the top, and the L1-S2 major groove base quadruple shown at the bottom. Nucleotide shading is the same as above.
Fig. 10
Fig. 10
Solution structural characteristics of the refined PEMV-1 P1-P2 pseudoknot (PDB 2RP0). A) Left, Global all atom superposition (residues 4–30) of 28 lowest energy models of the PEMV-1 pseudoknot (see Table 1 for structure statistics; right, ribbon representation of the same structure bundle color-coded as in Fig. 9. All-atom superposition above (B) and below (C) the helical junction of the PEMV-1 pseudoknot. Models of the BWYV (D), PLRV (E), PEMV (F) and ScYLV (G) pseudoknots emphasizing the structure of the minor-groove spanning L2 loop. The sequence (5′–3′) of L2 is also indicated below the figure.

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