Deciphering the influence of the [4Fe–4S] cluster of tRNA thiolation enzymes on tRNA binding

Monitoring of MmtRNA^Lys binding to apo- and holo-MmTtuI by fluorescence. (A) Fluorescence spectra obtained at λ_ex = 295 nm of apo-MmTtuI (0.5 µM) alone (in green), apo-MmTtuI + 3 mM AMP-CPP (in red), apo-MmTtuI + 2 µM tRNA^Lys (in blue), and apo-MmTtuI + 3 mM AMP-CPP + 2 µM tRNA^Lys (in magenta). (B) and (C) Saturation curves of W282 fluorescence at 355 nm normalized to tRNA^Lys concentration for apo-MmTtuI in the absence (B) and presence of 3 mM AMP-CPP (C). (D) Fluorescence spectra obtained at λ_ex = 295 nm of holo-MmTtuI (0.5 µM) alone (in green), holo-MmTtuI + 3 mM AMP-CPP (in red), holo-MmTtuI + 2 µM tRNA^Lys (in blue), holo-MmTtuI + 3 mM AMP-CPP + 2 µM tRNA^Lys (in magenta). (E) and (F) Normalized saturation curves of fluorescence at 355 nm, for holo-MmTtuI in the absence (E) and presence of 3 mM AMP-CPP (F).