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. 2019 Jun 27;8:e48385. doi: 10.7554/eLife.48385

Figure 2. Effect of C-terminal truncations on SecA function in vitro and in vivo.

(A) 900 nM Ru(bpy)2(dcbpy)-labelled SecA (Wild type; circles), SecAΔMBD (ΔMBD; triangles) or SecAΔCTT (ΔCTT; squares) was incubated in the presence of increasing concentrations of purified 70S ribosomes. Because error bars corresponding to one standard deviation obscured the symbols, they were omitted from the graph. The equilibrium dissociation constant (KD) of the complex was determined by fitting the increase in fluorescence anisotropy from the Ru(bpy)2(dcbpy) (lines; Table 1). (B) 0.5 μM SecA, SecAΔMBD or SecAΔCTT was incubated in the absence (lanes 1–3) of ribosomes, in the presence of 0.5 μM vacant 70S ribosomes (lanes 4–9) or in the presence of 0.5 μM RNCs containing nascent SecM peptide (lanes 10–12). Where indicated, binding reactions were incubated in the presence of 100 mM (lanes 1–6) or 250 mM (lanes 7–12) potassium acetate (KOAc). Binding reactions were layered on a 30% sucrose cushion and ribosomes were sedimented through the sucrose cushion by ultracentrifugation. Ribosomal pellets were resolved by SDS-PAGE and stained by Coomassie. (C) 600 nM IAANS-VipB peptide was incubated with increasing concentrations of SecA (Wild type; circles), SecAΔMBD (ΔMBD; triangles) or SecAΔCTT (ΔCTT; squares). Confidence intervals represent one standard deviation. The KD for the SecA-peptide complex was determined by fitting the increase in IAANS fluorescence upon binding to SecA (lines; Table 1). (D) Growth of strains producing SecA (DRH1119; bottom left), SecAΔMBD (DRH1120; bottom right) and SecAΔCTT (DRH1121; top) on LB plates containing 100 μM IPTG.

Figure 2.

Figure 2—figure supplement 1. CD spectra of SecA, SecAΔMBD and SecAΔCTT.

Figure 2—figure supplement 1.

Far-UV circular dichroism (CD) spectra of 2 µM solutions of SecA, SecAΔMBD, and SecAΔCTT in 10 mM potassium phosphate (pH 7.5).
Figure 2—figure supplement 2. Thermal denaturation plots of SecA, SecAΔMBD and SecAΔCTT.

Figure 2—figure supplement 2.

Representative plot of the thermal denaturation of SecA as determined by CD spectroscopy. The α-helical content of 2 µM solutions of SecA, SecAΔMBD, and SecAΔCTT in 10 mM potassium phosphate (pH 7.5) was determined by measuring molar ellipticity at 222 nm while the temperature of the solution was raised from 30°C to 50°C. The TMs listed in Table 1 were determined by van’t Hoff analysis.
Figure 2—figure supplement 3. Expression of SecA, SecAΔMBD and SecAΔCTT in strains DRH1119, DRH1120 and DRH1121.

Figure 2—figure supplement 3.

Strains DRH1119, DRH1120 and DRH1121 (relevant genotype: MC4100 ΔsecA attλ-placUV5-secA), which produce SecA, SecAΔMBD and SecAΔCTT, respectively, were grown in LB in the presence of 100 μM IPTG to mid-log phase. Cell lysates were normalised to cell density, resolved by SDS-PAGE and probed by western blotting using antisera against SecA and thioredoxin-1, as a loading control.