Figure 4.

Effect of the length of nascent peptide chain on mistranslocation event. (A) Peptide sequences used in this analysis. The N-terminal region of NlpC protein containing a PPP motif was fused with KKK and FLAG peptide. FLPL1: The complete N-terminal peptide sequence derived from NlpC. FLPL2–4: Shortened variants of FLPL1. (B) Translation yields of FLPL1–4 and RiP2 in titration of EF-G in the absence of EF-P. Peptides were labeled by [¹⁴C]-Asp and quantified by autoradiography. n = 3. (C) Ratios of full-length peptides FLPL1–4. The ratios were calculated as (yield of FLPL#)/[(yield of FLPL#) + (yields of RiP2)] using the values obtained in (B). n = 3. (D) Identification of the translation products by MALDI-TOF MS. Peptides were expressed with 0.03, 0.26 or 3.0 μM EF-G in the absence of EF-P for 20 min. Grey arrowheads: FLPL1–4, red arrowheads: RiP2, *: Na⁺ or K⁺ adduct peaks, †: unidentified peaks. +2-ME, +Cys and + DTT are 2-mercaptoethanol, cysteine and dithiothreitol adducts, respectively. ‘Calc.’ and ‘Obs.’ indicate calculated and observed m/z values, respectively. N.D.: not detected.