Figure 3.

Arrest-peptide-mediated force measurements upon formation of the ACTR-NCBD complex in vitro. (A) A schematic representation of the constructs used. ACTR (in light blue) is N-terminally fused to SecM sequences of increasing length (21–35 residues). Binding of fluorescently labeled NCBD creates force on the nascent chain that allows SecM stalling to be overcome and translation to continue to the C-terminal GFP. Similarly, NCBD was fused to SecM of increasing length (21–40 residues). (B) ACTR-SecM-GFP and NCBD-SecM-GFP constructs were translated in vitro, in the absence or presence of ∼8-μM CF680R-NCBD or ∼8-μM CF680R-ACTR, respectively. In-gel fluorescence recorded in the green (NCBD-SecM-GFP or ACTR-SecM-GFP) and red (CF680R-ACTR or CF680R-NCBD) channels is shown. (C) Quantification of the green fluorescent band intensities from the gels shown in (B) is depicted. (D) Difference between the expression of the ACTR-SecM-GFP constructs in the presence (+NCBD) and absence (-NCBD) of labeled NCBD, from experiments performed in triplicate, except from the construct ACTR-SecM-27, which was calculated from two experiments, is shown. To see this figure in color, go online. a.u., arbitrary units; Norm., normalized.