HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome

Mouzakis, Kathryn D.; Lang, Andrew L.; Vander Meulen, Kirk A.; Easterday, Preston D.; Butcher, Samuel E.

We utilized a well-established dual-luciferase frameshift assay (57, 67, 68) to quantitatively measure frameshift efficiency in RRL. The sequence of the frameshift site stemloop was varied to dissect the relative contributions of local and overall RNA stability on HIV-1 frameshift efficiency. We hypothesized that once the ribosome is paused on the slippery sequence, the thermodynamic stability of the base pairs encountered at the base of the stem-loop should be a critical determinant for frameshifting. After the ribosome has completed one translocation step, it moves away from the slippery site and the reading frame is established. Therefore, we further hypothesized that downstream base pairs in the stem-loop should have a much lower impact on frameshifting. To test this hypothesis, 12 MS constructs ( Figure 1B) were created using nearest-neighbor parameters (59) (60) (61) 69) to systematically alter the stability of different regions of the stem-loop. We define local stability (ÁG Local ) as the thermodynamic stability of base pairs directly downstream of the spacer ( Figure 1A) , as determined by their nearest-neighbor interactions (59) (60) (61) . Global stability (ÁG Global ) is defined as the overall thermodynamic stability of the stem-loop. The thermodynamic stabilities (ÁG Global ) were experimentally determined for a subset or RNAs (WT, MS1-2, MS5-7, MS10 and MS12) using UV-monitored thermal denaturation (Figure 2, Supplementary Figure S1 and Supplementary Table S2 ). Owing to the extreme stabilities of the structures (25), thermal denaturation curves were measured at low ionic strength (10 mM potassium phosphate buffer) in the presence of varying concentrations of urea and extrapolated back to 0 M urea (58) . Results followed the same trend as those predicted from nearest-neighbor parameters (59-61,69) (R 2 = 0.95) ( Figure 2 ). As expected, the measured stabilities were lower than the predicted values at 1 M NaCl (70) . Upon correction of the experimental values to 1 M monovalent ionic strength (71), we observe an excellent agreement (R 2 = 0.95) between experimental and predicted free energies ( Figure 2) . Indeed, free energy prediction is robust for small, stable RNAs with no competing suboptimal folds (20) .