RelE cleavage of mRNA in the A site of ribosomal complexes. (a) The mRNA fragments resulting from RelE cleavage in the A site of the three ribosomal complexes Init (see Figure 1a), preT (see Figure 1b) and postT (see Figure 1c), separated on a 10% sequencing gel. The amount of radioactivity in the postT lane was doubled to make the AUU cleavage visible. (b) Time-dependent cleavage of mRNA by RelE; preT ribosomes were incubated with EF-G together with GDPNP (+ GDPNP 2) or GTP (+ GTP) or GDP (+ GDP). RelE was added after 10, 25 or 40 min, and the reaction was in each case quenched 5 min after RelE addition. Alternatively, preT ribosomes were incubated together with EF-G, RelE and GDPNP and the reaction quenched after 15, 30 or 45 min (+ GDPNP 1). (c) Time-dependent cleavage of mRNA by 120 nM RelE in the A site of 0.3 μM postT or preT ribosome complexes incubated with 2 μM EF-G and 0.6 mM GDPNP. As a control, in the last two lanes 1 mM GTP was added to postT or preT ribosomes at the end of the incubation.