Fig. 2
From: Hierarchical mechanism of amino acid sensing by the T-box riboswitch
smFRET and kinetic analysis of tRNA binding to Stem-I. a Example smFRET trace of Cy5-labeled tRNAGly binding to Stem-I hybridized with Cy3-labeled DNA oligonucleotide at the 3′-end. Green, Cy3 intensity; red, Cy5 intensity; black, FRET efficiency. b Population FRET histogram generated from the binding events and fit with a single Gaussian function (black). The mean FRET value given by the center of the peak provides an estimate of the distance between the fluorophores, which in this example is between the base of Stem-I and U46 of the bound tRNA. c Example binding trace showing Cy3 intensity that photobleaches in a single-step indicative of one T-box riboswitch molecule, and abrupt increases in Cy5 intensity, indicative of tRNA binding and dissociation events from which dwell times in bound and unbound states, tbound and tunbound, can be obtained. d Representative single-molecule traces showing tRNA binding to Stem-I. The Cy5 signal is shown in black and a two-state hidden Markov model (HMM) idealized trace is shown on top in red. Dwell-time histograms for e tunbound and f tbound were fit with single-exponential functions (blue and red), to estimate the rate constants, kon and koff, respectively. Errors are s.d. of at least three independent replicates
