Figure 1.
Deletion analysis identifies the minimal attenuator element in SARS-CoV viral sequence.
(A) Schematic drawing of SARS-CoV viral genomic region spanning−1 PRF signal and its upstream element covering sequences 13222–13520 of SARS-CoV. Each of different 5′-viral deletion fragments (annotated by arrow) was cloned into a dual-luciferase−1 PRF reporter with a shortened −1 reading-frame. (B) In vitro −1 PRF assays by SDS-PAGE analysis of 35S methionine-labeled translation products for reporter constructs containing different 5′ deletions of the ATT element (left) and the relative frameshifting activities of different deletion mutants (right). The two major bands in each lane correspond to 0 (lower) and −1 (higher) frame translation products. The relative extent of frameshifting was determined as the ratio between each mutant and the 13390–13520 containing reporter construct (being treated as 100%). The value for each construct is presented as mean±SD (error bars) of triplicate experiments.
Figure 2.
Base-pairing formation of the predicted hairpin stem is required for efficient −1 PRF attenuation activity.
(A) Illustration of constructs with different base-pairing schemes at the lower stem of the predicted hairpin. For each mutant, the nucleotide composition after mutation is boxed or boldly typed. Two nucleotides, 27 nucleotides upstream of the 0-frame E site and those involved in terminal stem base-pairing formation, are colored for comparison. (B) In vitro −1 PRF assays by SDS-PAGE analysis of 35S methionine-labeled translation products for constructs with different base-pairing schemes. The 0 and −1 frame products are labeled as indicated. (C) Relative frameshifting activity of (B) with the frameshifting efficiency of construct 13390–13520 as 100% (for comparison purposes). Value for each construct was the mean of three independent experiments, with the bar representing the standard error of the mean.
Figure 3.
Sequence composition of the predicted hairpin stem is crucial for its −1 PRF attenuation activity.
(A) The scheme for swapping the base-pairing composition of the attenuation hairpin stem in constructs containing a longer SARS-CoV viral sequence. The covered viral sequences (13318 to 13391) and the predicted secondary structure are shown, with the six swapped base pairs boxed for comparison, while the slippery site is underlined and followed by a SARS-CoV pseudoknot (SARS-PK) stimulator. (B) In vitro −1 PRF assays by SDS-PAGE analysis of 35S methionine-labeled translation products for constructs in (A) (left), and the relative frameshifting activity with that of 13318-WT being treated as 100% (right). Error bars, s.d.; n = 3. (C) Relative frameshifting activity calculated from dual-luciferase assay data using 293T cells harboring transiently expressed 13318-WT and 13318-6BPGC constructs with the frameshifting efficiency of 13318-WT being treated as 100%. Error bars, s.d.; n = 3.
Figure 4.
The UGCG loop and G bulge are not the major determinants for −1 PRF attenuation whereas the attenuation efficiency of a hairpin is positively correlated with its stability.
(A) Illustration of constructs with G-bulge or UGCG loop (shaded in green) disruption and with 5′-half sequence deletion (boxed) of the lower stem of the predicted hairpin. The bulge G was converted into a GC base pair by inserting a C in its complementary strand. An upstream C nucleotide was deleted and accompanied with an A to G replacement to correct the frame and prevent an in-frame stop codon, respectively. The modified sequences in AddLoop and rBulge constructs are typed in blue and red, respectively. (B) In vitro −1 PRF assays by SDS-PAGE analysis of 35S methionine-labeled translation products for constructs in (A). The 0 and −1 frame products are labeled as indicated. (C) Relative frameshifting activity of (B). Frameshifting efficiency of the construct 13318–13520 was treated as 100% for comparison. Value for each construct was the mean of three independent experiments, with the bar representing the standard error of the mean. (D) Correlation between attenuation efficiencies (against the 13390–13520 construct) and predicted free energy values of the 6GC-hairpin variants in Fig. S2. A linear regression line is shown with the equation for the line and the regression statistic, R2, and a threshold stability below which the hairpin does not attenuate frameshifting. Attenuation efficiency was calculated according to the definition given in experimental procedures.
Figure 5.
Proximity to the slippery site determines the attenuation potency of a hairpin.
(A) Scheme showing the inserted nucleotide sequences between the extended base of the 6BPGC hairpin stem and the slippery site with spacing numbers after insertion shown in parentheses. The two extended base pairs involving spacer are boxed. The inserted sequences were designed to retain no stable secondary structure and regenerate the 3′-flanking CGUU sequence to prevent flanking sequence effects affecting frameshifting efficiency [12], [13]. (B) In vitro −1 PRF assays by SDS-PAGE analysis of 35S methionine-labeled translation products for constructs with insertions as shown in (A). (C) Relative frameshifting activity with that of the construct 13390–13520 being treated as 100%. Error bars, s.d.; n = 3. (D) Relative −1 frameshifting activity based on dual-luciferase assays from yeast cells, transfected with reporters containing selected insertion mutants in (A) and SARS-PK replaced by DU177 pseudoknot. Frameshifting efficiency of the construct containing the DU177 pseudoknot alone was treated as 100% (for comparison purposes). Error bars, s.d.; n = 3. (E) In vitro −1 PRF assays by SDS-PAGE analysis for reporter constructs (with or without nucleotide insertion in the region between the extended base of 6BPGC hairpin stem and the slippery site) under different conditions with variations in relative amounts of mRNA and Retic lysate. Condition designations: 2M1R (mRNA 100 ng/Retic lysate 1.7 μl); 1M1R (mRNA 50 ng/Retic lysate 1.7 μl); 1M2R (mRNA 50 ng/Retic lysate 3.4 μl) in a total of 5 μl/reaction. (F) Relative frameshifting activity of (E) using frameshifting efficiency of 13390–13520 construct in 2M1R condition as 100%. Error bars, s.d.; n = 3.
Figure 6.
Effects of E Site sequences and downstream pseudoknot stimulator identities on the attenuation activity of M1 hairpin.
(A) The sequences of M1 attenuator hairpin with the mutation site boxed and 5′WT-M1 element with the mutation sites boldly typed.(B) The sequence variation (top), SDS PAGE result of −1 PRF assays (middle) and attenuation efficiencies of M1 attenuator (bottom) of reporter constructs with E site sequence variation. Error bars, s.d.; n = 3. (C) The −1 PRF module set-up (top), SDS PAGE result of −1 PRF assays (middle), and attenuation efficiencies of M1 attenuator (bottom) of reporter constructs with different downstream pseudoknot stimulators. Δ means the attenuator hairpin is deleted. The 0 frame and −1 frame products are designated by filled circles and triangles, respectively. Error bars, s.d.; n = 3.
Figure 7.
The 6BPGC hairpin serves as a +1 PRF stimulator when placed upstream of a +1 shifty site in yeast.
(A) Schematic drawing for the flanking sequences surrounding the 6BPGC hairpin in yeast +1 frameshifting reporter constructs. Construct with a disrupted attenuator hairpin (5′-WT) was designated by Δ and the 5′-flanking sequence of 6BPGC hairpin was designed to prevent forming base-pairs with E site sequences. (B) Fold change of the AGG- and 6BPGC-dependent +1 frameshifting activity in yeast using the activity of the CGG.E.CGC construct as 1. Error bars, s.d.; n = 6.