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. 2016 Apr 6;113(16):E2286–E2295. doi: 10.1073/pnas.1521156113

Fig. S5.

Fig. S5.

Effect of GE81112 on the rate of exchange between 30S-bound and free IF1 and IF2 and on the kinetics of mRNA binding to the 30S subunit. (A and B) Two types of complexes were prepared in the presence (red tracings) and absence (black tracings) of GE81112. One complex contained fluorescently labeled IF1 4-ALEXA555 and fluorescein-labeled fMet–tRNA; IF1 dissociation is monitored by the reduction of the FRET signal between the two fluorescent ligands upon the addition of a 10-fold excess of nonfluorescent IF1 (A). The other complex contained fluorescently labeled IF2 757-Alexa488 and fMet–RNA8-QSY35 (B). Upon the addition of a 10-fold excess of nonfluorescent factor, the dissociation of IF1 (A) causes a decrease in fluorescence intensity (because of the loss of the FRET signal), whereas IF2 dissociation (B) causes an increase in fluorescence because of diminished quenching. (C and D) Fluorescence stopped-flow binding kinetics of 3′ fluorescein semithiocarbazide-labeled (24) leadered (C) and leaderless (D) mRNA to 30S subunits (black and red tracings) or to 30S subunits containing all components of the initiation complex except for the mRNA (green and blue tracings) in the absence (black and green tracings) or in the presence (red and blue tracings) of 100 µM GE81112.