Figure 8.
Effect of alterations to the four-way junction on pseudoknot folding. (A) Diagram of the expected tertiary structures of the four-way junction of wild-type, S3mut (A51C/G65C), 8U insert, and 4U insert mutations. Red lines indicate the position of the 8- and 4-nucleotide poly-uridine insertions replacing residues 51–70 of the DB RNA. (B) smFRET histograms of the four constructs in conditions containing 1 mM MgCl2. FRET values of 3289, 7293, 4092 and 5661 molecules respectively were collected over 2 seconds at a 0.1 s integration time to generate histograms of all FRET values observed. Histograms were fit to the sum of two Gaussian distributions, with fits displayed on graph as well as the relative percentage of the ∼0.5 and ∼0.8 FRET states. (C) Effect of MgCl2 on pseudoknot folding of Wt, S3mut, 8U insert, and 4U insert constructs. smFRET histograms (Supplementary Figure S14) were collected as in (B). The fraction folded in the pseudoknot (∼0.8 FRET state) were plotted against the MgCl2 concentration in the experiment.