(A) SDS-PAGE and western blot (against His6) analysis of purified Get3•TA complexes. Get3•TA complexes were assembled during in vitro translation in S30 extract in the presence of Sgt2, Get4/5 and Get3, and purified via the 3xStrep-tag on the TA protein as described in the Materials and methods. T7 RNA polymerase, Sgt2, Get3, and Get4 were His6-tagged. I denotes input onto the Strep-Tactin resin, E denotes elution. (B) Representative autoradiograms of insertion reactions. The asterisk (*) denotes a truncated translation product of the TA, which was inserted and glycosylated (**) with similar efficiencies as the full-length translation product. (C) Quantification of the insertion reactions in panel B. The data were fit to Equation 5, which (together with replicates) gave observed insertion rate constants (kobsd) of 0.14 ± 0.01, 0.048 ± 0.006, and 0.041 ± 0.003 min−1, and translocation end points (T) of 64 ± 8, 63 ± 1, and 61 ± 2% for Bos1, Bos1-FisC, and Bos1-RRRR, respectively. (D) Summary of the rate constants of competing molecular events, defined in Figure 7F and Equations 6–7, that contribute to the observed targeting/insertion reactions in panel C. Values represent mean ± S.E.M., with n = 2. (E) Targeting and insertion of Get3•Bos1 and Get3•Bos1-FisC complexes in the absence of nucleotides.