Stem-loop structures can effectively substitute for an RNA pseudoknot in -1 ribosomal frameshifting

Abstract

-1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base-base and base-sugar interactions between loop and stem nucleotides. Recently, we showed that mutation of bases involved in triple helix formation affected frameshifting, again emphasizing the role of the triple helix in -1 PRF. Here, we investigated the efficiency of hairpins of similar base pair composition as the SRV-1 gag-pro pseudoknot. Although not capable of triple helix formation they proved worthy stimulators of frameshifting. Subsequent investigation of ∼30 different hairpin constructs revealed that next to thermodynamic stability, loop size and composition and stem irregularities can influence frameshifting. Interestingly, hairpins carrying the stable GAAA tetraloop were significantly less shifty than other hairpins, including those with a UUCG motif. The data are discussed in relation to natural shifty hairpins.

Figure 1.

Hairpin derivative of the Simian retrovirus type-1 (SRV-1) gag-pro frameshift pseudoknot is an efficient frameshift stimulator. (A) Schematic representation of the SRV-1 pseudoknot (SRV-pk) and its hairpin derivative (SRV-hp). Mutations in SRV-pk loop L2 (SRV-mutpk) and SRV-hp (SRV-muthp) are indicated. The slippery sequence is underlined. (B) SDS–PAGE analysis of ³⁵S-methionine-labeled translation products in rabbit reticulocyte lysate (RRL). −1 Ribosomal frameshifting is monitored by appearance of the 65-kD product (FS). The non-shifted zero-frame product is indicated by NFS. Quantitative analysis of frameshifting efficiency [FS (%)] is described in ‘Materials and Methods’ section.

Figure 2.

Influence of stem length of UUCG-capped hairpins on −1 ribosomal frameshifting efficiency in vitro and in vivo. (A) SDS–PAGE analysis of ³⁵S-methionine-labeled translation products in RRL using mRNAs with hairpins of various stem lengths. See legend to Figure 1B for more details. The base composition of the various stems is shown. The 0 bp is the control without a hairpin. (B) Graph showing the relation between frameshifting efficiency (indicated by bars) on the left y-axis and predicted thermodynamic stability by MFOLD [indicated by a solid diamond (filled diamond) on the right y-axis]. The average FS (%) and error bars were from at least three independent experiments. (C) Selected hairpins with different stem lengths were assayed for their ability to induce −1 ribosomal frameshifting in HeLa cells. The frameshifting efficiency was obtained by measuring dual-luciferase activity of a frameshift reporter construct (see ‘Materials and Methods’ section). The in vivo experiments were done at least three times in triplicate.

Figure 3.

Influence of bulges and mismatches in the middle part of the hairpin on frameshifting efficiency. (A) SDS–PAGE analysis of ³⁵S-methionine-labeled translation products in RRL using mRNAs containing the indicated hairpins. See legend to Figure 1b for more details. (B) Graph showing the relation between the predicted thermodynamic stability and frameshift efficiency. See legend to Figure 2B for more details.

Figure 4.

Influence of loop sequence and closing base pair (cbp) on −1 ribosomal frameshifting efficiency. The composition of various loops capping a 9 bp stem is shown in bold, and CG-cbps are shown in lower case. The constructs are named after their loop sequence followed by the ‘/cg’ extension when the cbp was changed from G–C to C–G. Slippery sequence and spacer are the same as in the construct shown in Figure 1A. Graph is similar to that of Figure 2B except that on the right y-axis ΔG starts from −18 kcal/mol.

Figure 5.

Comparison of in vitro and in vivo frameshifting efficiencies induced by four selected 9 bp hairpins with different loops. (A) SDS–PAGE analysis of ³⁵S-methionine-labeled translation products in RRL of mRNAs containing the 9 bp hairpin with the indicated loop sequence. See legend to Figure 1B for more details. (B) Comparison of −1 ribosomal frameshifting in vitro and in vivo. The in vitro efficiency (white bar) was obtained by quantifying autoradiograms and averaging of at least three independent experiments. In vivo frameshifting efficiency (black bar) was obtained by measuring dual-luciferase activity of a frameshift reporter construct in HeLa cells (see ‘Materials and Methods’ section). The in vivo experiments were done at least three times in triplicate.

Figure 6.

Overview of naturally occurring hairpins involved in −1 ribosomal frameshifting and two synthetic pseudoknot-derived hairpins (SRV and IBV). HIV, Human immunodeficiency virus type 1 gag-pol; HTLV, Human T-lymphotropic virus type 2 gag-pro; CfMV, Cocksfoot mottle virus; IBV, Infectious bronchitis virus; SRV, Simian retrovirus type 1.