Figure 3.
Stability of 50S subunits with A loop mutations at positions 2554–2555. (A) In the HiBit assay, the 12 amino acid HiBit peptide is translated by the ribosome, which activates a truncated nanoluciferase (11S). 50S subunits were pre-incubated at 37°C or 60°C and then their relative activities were determined with the HiBit assay (data is represented as the mean of three replicates). Both untagged and MS2-tagged WT 50S subunits were used as controls. Error bars represent the standard deviation of three independent reactions. (B) 50S subunits were heat treated at 37°C (black dash) or 60°C (red) and then resolved on a 15–40% sucrose gradient. (C) RNA oligonucleotides (15 nucleotides) containing the WT A loop sequence (2547–2561), the CU or CC mutant sequences were melted on a CD spectrometer. The melting temperature (Tm) was calculated for each construct using a two-state model.