Figure 1
Ternary complex formation with RCys-tRNA and incorporation of RCys into dipeptides. (a) Chemical structures of fluorescent probes covalently attached to the amino acid side-chain thiol group to form RCys-tRNACys. (b) TC formation with RCys-tRNACys (10 nM) upon addition of increasing concentrations of EF-Tu–GTP monitored by the fluorescence change of the R probe (Figure S2). Data were normalized by setting the fluorescence intensity of RCys-tRNACys in the absence of EF-Tu–GTP to 0 and the saturating fluorescence intensity extrapolated by hyperbolic fitting to 1. Lines represent hyperbolic fits using a one site binding model (Material and Methods). The Kd value is reported with the standard error of the fit. (c) Formation of f[3H]Met-RCys dipeptides on 70S IC (20 nM) in the presence of RCys-tRNACys (0.12 µM), EF-Tu–GTP (75 µM), and increasing concentration of E. coli aa-tRNA without Cys-tRNACys. f[3H]Met-RCys peptides were separated by HPLC (Figure S3). Lines represent fits to a competition model yielding [TC(aa)]0.5 as the concentration of TC required to reduce dipeptide formation by half of 4.5 ± 0.6 µM , 5.2 ± 0.4 µM, and 4.5 ± 0.4 µM for NBD, BodipyFL, and Atto520, respectively. The selectivity S for the RCys-tRNACys relative to near-cognate aa-tRNAs indicated in the Figure was calculated taking into account the abundance of the near-cognate aa-tRNAs in the total tRNA pool (Methods). For the translation experiments in the next sections [TC(aa)] = 30 µM was kept constant (boxed).
