Preparation of Mutant FtsY Proteins

Plasmids for mutant FtsY were constructed using the QuikChange mutagenesis protocol (Stratagene). The expression construct for His₆-FtsY-dN1 was a kind gift from the Schaffitzel Lab. mRNAs for in vitro translations were synthesized by in vitro transcription using T7 (for RNC preparation) or SP6 (for co-translational translocation assay) polymerases following the Megascript protocol (Ambion). Wildtype and mutant Ffh and FtsY and 4.5S RNA were expressed and purified as described in previous studies (Peluso et al., 2001 (link); Lam et al., 2010 (link)). RNCs bearing signal sequences of FtsQ or Luc were prepared as described previously (Zhang et al., 2010 (link)). FtsY-C345 and Ffh-C153 were labeled with Alexa647-maleimide (Thermo Fischer, Waltham, MA) and FtsY-C356 was labeled with acrylodan (Invitrogen) as described (Shen et al., 2012 (link)) with the minor modifications. Details see the SI Methods. All proteins were exchanged into SRP buffer (50 mM HEPES-KOH, pH 7.5, 150 mM KOAc, 10 mM Mg(OAc)₂, 2 mM dithiothreitol (DTT) and 0.01% octaethyleneglycol dodecylether (Nikkol)) prior to use.