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.

For the WT and a subset of the mutant stem-loop (MS) RNAs (Supplementary Table S2 and Figure 2 ), RNA overall thermodynamic stability, ÁG Global , was measured using UV absorbance at 260 nm as a function of temperature with a Cary Model 400 Bio UV-visible spectrophotometer equipped with a Peltier heating accessory and temperature probe. All samples contained 10 mM potassium phosphate buffer, pH 7.0, 2 mM RNA, in a volume of 1 ml. For RNAs that were too stable to measure ÁG Global under these conditions, urea was added to 4, 6 and 8 M, and the ÁG Global was deduced by extrapolating to 0 M urea as described below. Prior to data collection, samples were heated from 20 C to 95 C, at 10 C/min, held at 95 C for 5 min and cooled from 95 C back to 20 C at the same rate to ensure homogenous folding. Samples were heated at 1 C/min from 20 C to 95 C. Identical traces were obtained by cooling, indicating a lack of hysteresis. A 260 data were collected in 0.5-min intervals and raw data were baseline corrected by subtraction of A 320 values at each temperature. The average hyperchromicity [Equation (1) ] and temperature were calculated from four curves and the SEM was determined for each average.