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. 2016 Jun 2;5:e13429. doi: 10.7554/eLife.13429

Figure 5. Tetrapeptide translocation (Step 12) is faster than tripeptide translocation (Step 9).

(A) Puromycin reaction with PheValLys-tRNALys bound either at the A site (D) or at the P-site (O) of the 80S·FVKM-IRES complex or being translocated from the A site to the P site (□). (B) Puromycin reaction with PheValLysMet-tRNAMet either bound at the P-site (O) of the 80S·FVKM-IRES complex or being translocated from the A site to the P site (□). Lines in A. and B. Are fits to single exponentials. (C) Time dependence of PheLysValArgGlnTrpLeuMet octapeptide synthesis from the 80S·FKVRQWLM-IRES complex containing various peptidyl-tRNAs pre-bound at the P site, as indicated. The pre-bound peptidyl tRNAs were prepared using the standard procedure (see Complex Preparations in Materials and methods) by incubating the 80S-IRES complex with the relevant TCs for 15 min. The remaining TCs needed for octapeptide synthesis, including [35S]-Met-TC, were then added, each at a concentration of 1.6 µM, for the indicated times prior to quenching. PheLysValArgGlnTrpLeuMet octapeptide synthesis was measured by [35S]-Met cosedimenting with 80S ribosome.

DOI: http://dx.doi.org/10.7554/eLife.13429.014

Figure 5.

Figure 5—figure supplement 1. Octapeptide synthesis: 80S·FKVRQWLM-IRES complex with FKVRQWLM-tRNAMet in the P-site was prepared using the standard procedure (see Complex Preparations in Materials and methods) and incubating the 80S-IRES complex with the eight relevant TCs (including [35S]-Met-TC) for 40 min.

Figure 5—figure supplement 1.

The resulting labeled octapeptide, released by base hydrolysis, was analyzed by TLE. Migration positions of [35S]-Met and [35S]-labeled FKVRQWLM (*) are indicated.