Reactivity of pre-translocation-state ribosome complex I. (A) Complex I was prepared by successive binding of uncharged tRNA^Met and N-Acetyl-Phe-tRNA^Phe to ribosomes programmed with a gene 32 mRNA variant with AUG UUC UUC (MFF) as the first three codons in its open reading frame. (B) Primer extension analysis of complex I before and after incubation with EF-G and GTP. Ribosome complexes were formed on gene 32 variant mRNA pre-annealed to a [³²P]-labeled primer. Markers include tRNA^Met (lane 1) which yields a band at position 0, and tRNA^Phe (lane 2) which yields a band at position +3. The pre-translocation-state complex (lane 3) has a diagnostic pattern with a doublet heavier at the +1 position. Movement to the +3 position is promoted by EF-G (lane 4). (C) Puromycin reactivity of complex I. Complex I was incubated with 10 mM puromycin at 37°C (open squares), and the fraction N-Ac-Phe converted to N-Ac-Phe-Pm is shown on the y-axis. Puromycin reactivity of the corresponding post-translocation complex (filled squares), formed after incubating complex I with 5 μM EF-G for 5 min at 37°C, is also shown.

Stability of pre-translocation ribosome complex II. (A) The pre-translocation complex II was incubated at 37°C for 2 (open circles), 10 (filled diamonds) or 45 (open squares) min, before starting the reaction with the addition of puromycin (1 mM). The fraction of fMet-Phe converted to fMet-Phe-Pm was determined where the time of addition of puromycin is taken as time zero in each case. As a control, puromycin reactivity of the post-translocation complex (filled squares) was determined after incubating complex II with 5 μM EF-G (as in Fig. 2 ▶). (B) Chase experiment performed with N-Ac-Phe-tRNA^Phe with pre-translocation-state complex II. Reaction was started with the simultaneous addition of 4 equivalents of N-Ac-Phe-tRNA^Phe (1 μM final) and 10 mM puromycin, and time points were taken. The fraction of fMet-Phe converted to fMet-Phe-Pm was determined in the absence or presence (filled or open symbols, respectively) of tRNA competitor in buffer B at 7 mM or 15 mM MgCl₂ (circles or squares, respectively).

Compensatory base analysis of G2553 mutant ribosomes with C75 tRNA mutant A-site substrates in complex II. The rate of reactivity of the wild-type and mutant ribosome complexes with 8 μM CpPm and GpPm was determined (Kim and Green 1999).

Model for sampling of intermediate (hybrid) tRNA binding states by pre-translocation-state bound N-Ac-Phe-tRNA^Phe and fMet-Phe-tRNA^Phe, where the dipeptidyl tRNA equilibrium favors hybrid-state tRNA binding compared with the equilibrium for N-Acetyl-Phe-tRNA^Phe.

Reactivity of pre-translocation-state ribosome complex II. (A) Complex II was prepared by successive factor-dependent binding of fMet-tRNA^Met and Phe-tRNA^Phe to the P and A sites of ribosomes programmed with gene 32 variant mRNA (as above) where peptide-bond formation generates deacylated and dipeptidyl tRNA species. (B) Primer extension analysis of complex II before and after incubation with EF-G and GTP. Markers include tRNA^Met (lane 1) which yields a band at position 0, and tRNA^Phe (lane 2) which yields a band at position +3. The pre-translocation-state complex (lane 3) has a diagnostic pattern with a doublet heavier at the +1 position. Movement to the +3 position is promoted by EF-G (lane 4). (C) Complex II was incubated with equimolar Phe-tRNA^Phe in complex with EF-Tu/GTP before (open squares) and after (filled squares) translocation with 5 μM EF-G (as above). The fraction of fMet-Phe converted to fMet-Phe-Phe is shown on the y-axis. (D) Puromycin reactivity of complex II in buffer B with 7 mM MgCl₂. Complex II was incubated with 10 mM puromycin, and the fraction of fMet-Phe converted to fMet-Phe-Pm was determined before (open squares) and after (filled squares) translocation with 5 μM EF-G (as above). (E) Puromycin reactivity of complex II in polymix buffer (Jelenc and Kurland 1979). Complex II was incubated with 10 mM puromycin, and the fraction of fMet-Phe converted to fMet-Phe-Pm was determined before (open squares) and after (filled squares) translocation with 5 μM EF-G (as above).

Primer extension analysis (toeprinting) of complex II before and after reaction with puromycin. Ribosome complexes were formed on gene 32 variant mRNA pre-annealed to a [³²P]-labeled primer. As controls, the first and second codons in the gene 32 variant open reading frame were bound by their cognate tRNAs, fMettRNA^Met (lane 1) or Phe-tRNA^Phe (lane 2), alone before carrying out the primer extension reaction. Lanes 3 and 4: The toeprint for complex II, before and after reaction with puromycin, respectively. Lanes 5 and 6: The toeprint for complex II, before and after reaction with puromycin, and after incubation with 5 μM EF-G (as above).

Dependence of pre-translocation-state puromycin reactivity on puromycin concentration. The time courses of fMet-Phe-Pm formation by complex II were fitted to single exponential rate equations in order to obtain the k_obs for each puromycin concentration in buffer B with either 7 mM or 15 mM MgCl₂. Fitting the data to the Michaelis-Menten equation gave a K_1/2 of 5.9 or 10.3 mM and a k_cat of 0.024 or 0.014 s⁻¹.