Fig. 3. The TM sequence of FtsQ RNCs interacts sequentially with SecY and YidC. (A) In vitro synthesis of nascent FtsQ 70mer, 77mer, 87mer, 97mer and 108mer (all with a TAG codon at position 40) was carried out in the presence of IMVs and (Tmd)Phe-tRNASup. Aliquots were TCA-precipitated directly to evaluate translation and suppression efficiencies (not shown). After translation, samples were kept in the dark or UV-irradiated and subsequently extracted with carbonate. UV-irradiated pellet fractions were immunoprecipitated using antiserum against SecY or YidC. Immunoprecipitated adducts obtained with 87FtsQTAG40 are shown (lanes 11 and 12). The translation product at 35–40 kDa, present in lanes 1–10, is the peptidyl-tRNA form of the nascent FtsQ. SecY adducts are indicated by an open square; YidC adducts are indicated by an asterisk. (B) Nascent chains present in carbonate pellets from non-irradiated samples (A, lanes 1–5) were quantified. Carbonate resistance was quantified and expressed relative to the total amount of suppressed nascent chains present in TCA-precipitated translation totals (not shown). (C) SecY and YidC crosslinking adducts from (A) (lanes 6–10) were quantified and each signal was corrected for the total amount of carbonate-resistant material. Highest values for crosslinking efficiency were taken as 100%. The quantification is the average of two independent experiments. Hatched boxes, SecY adduct; black boxes, YidC adduct. For details on the quantification procedure see Supplementary data. (D) FtsQ nascent chains were produced and crosslinked as described in (A) and analyzed by tricine SDS–PAGE. To identify lipid crosslinking adducts (indicated by an ‘o’), photo-crosslinked 77FtsQTAG40 nascent chains were incubated with bee venom phospholipase (PLA2).