FIGURE 1.

TF interactions with nascent chains during translation in real time. A, crystal structure of E. coli TF (PDB code 1w26), with the N-terminal domain shown in red, the PPIase domain in yellow, and arm 1 and arm 2 of the C-terminal domain in green and blue, respectively. Engineered cysteines to which the NBD or BADAN probes were covalently bound are indicated by black circles and their amino acid number. B, determination of the specific activity of Luc in the absence (control) or presence of 5 μm WT TF, TF150-NBD, TF326-NBD, or TF376-NBD. The specific activity of Luc upon translation in the PURE system was normalized with respect to the control (set to 1). Standard deviations from three independent experiments are shown. C, in vitro translation of Luc in the PURE system in the presence of either 1 μm TF326-NBD (green) or TF(FRK/AAA)326-NBD (purple) and translation of α-Syn (gray) in the presence of TF326-NBD. D, in vitro translation of Luc in the presence of either 1 μm TF-B (green) or TF(FRK/AAA)-B (purple) and translation of α-Syn (gray) in the presence of TF-B. Translations were initiated by the addition of the respective DNA templates and performed at 30 °C. E, a representative autoradiograph of Luc and α-Syn translation kinetics during a real-time experiment with aliquots taken at various time points as indicated.