Localized ribosome access and distal tuning via the Listeria prfA RNA thermometer

Figures

• 

  Figure 1.. Compaction of the prfA RNAT upon addition of magnesium. (A) SV-AUC Mg2+ titrations reveal a reduction in frictional ratio, indicative of compaction of the RNA into a more spherical conformation. Red, yellow, and blue data points highlight 100 nM, 1 mM and 20 mM Mg2+, respectively. (B) Calculated radius of hydration (Rh) at 100 nM, 1 mM and 20 mM Mg2+ demonstrate compaction across the titration. (C) A pseudo 3D overlay of the Monte Carlo Genetic Algorithm results at the indicated Mg2+ concentrations.

• 

  Figure 2.. Competitive SiM-KARTS analysis of ribosome binding site (RBS) conformational dynamics. (A) Schematic of the SiM-KARTS construct showing the localization probe, laser excitation wavelengths and immobilization via biotin-streptavidin (B and SA) interaction on a biotin-PEG passivated cover slide. SiM-KARTS is total internal reflection technique, producing an evanescent field which excites fluorophore-labeled probes only when near the immobilized RNA. (B) Representative traces of the Cy5-labeled RBS probe in the absence (top) and presence of the dark probe (bottom). The binding frequency is greatly reduced as indicated by HMM idealization. (C) Kinetics analysis of the competitive SiM-KARTS experiment demonstrates reduced binding kinetics due to the presence of the unlabeled competitor probe for the RBS region. Statistical analysis was performed using Welch and Brown-Forsythe ANOVA tests. The data represent the kinetic model derived from a minimum 3 independent trials and shows the standard deviation.

• 

  Figure 3.. Probing the local dynamics of the prfA RNAT via SiM-KARTS. (A) Schematic of the SiM-KARTS construct. Independent experiments were conducted with a probe for either the RBS or H4 region. (B) Example SiM-KART traces for the RBS probe (top) and the H4 probe (bottom) at both 30 °C and 37 °C. HMM idealizations are shown above their respective traces. (C) The relative change in RBS (orange) and H4 (green) probe accessibility, normalized to the 30 °C condition for each probe. The H4 probe binding kinetics remain relatively constant, indicative of a well folded structure, however the RBS probe binding kinetics increase by a factor of 4 across the assayed temperatures. This change in accessibility indicates a loss of secondary structure in the region around the RBS. Statistical analysis was performed using Tukey multiple comparison with the two-way ANOVA. Data represent the kinetic model derived from a minimum 3 independent trials.

• 

  Figure 4.. In vitro transcription/translation analysis reveals the temperature sensitivity of the prfA RNAT. (A) A schematic of the in vitro transcription/translation construct and labeled tRNA-Lys. A plasmid containing the prfA RNAT and PrfA coding sequence or the DHFR gene is added to the kit components and supplemented with fluorescently labeled tRNA-Lys to facilitate gel-based quantification of translation products. (B) Representative translation gel comparing the non-thermal regulated translation of DHFR against the prfA RNAT-regulated PrfA. (C) Quantification of in vitro translation results comparing DHFR to PrfA. The prfA construct demonstrates strong translation activation at 34 °C. The results represent the mean and standard deviation of 3 independent replicates.

• 

  Figure 5.. Mutations within the prfA RNAT alter RNA structure and impact temperature-regulated translation. (A) Secondary structure of the prfA RNAT (center) with the destabilizing (H4, left) and stabilizing (L5, right) mutations. (B) SiM-KARTS construct where the prfA-WT, -H4, and L5 RNATs were interrogated with RBS and H4 probes. (C) Example SiM-KART traces for prfA-H4 (top) and prfA-WT (bottom) with the H4 probe at 37 °C. HMM idealizations are shown above their respective traces. (D) Quantification of the H4 probe binding kinetics for prfA-H4 and prfA-WT revealed a higher frequency of binding to the prfA-H4 RNAT due to the destabilizing mutations. Data represents the derived kinetic models from a minimum of 3 replicates. Welch’s t test was used for statistical analysis. (E) Example SiM-KART traces for prfA-H4 (top) and prfA-WT (middle), and prfA-L5 (bottom) with the RBS probe at 37 °C. HMM idealizations are shown above their respective traces. (F) Quantification of the RBS probe binding kinetics for prfA-RNAT mutants revealed that distal mutations alter RBS accessibility. Data represent the kinetic models derived from a minimum of 3 independent experiments. A Welch and Brown-Forsythe…