Effect of RRF on the release of inorganic phosphate from ribosome-bound EF-G. (A) Effect of RRF on the delay of Pi release. Post-termination complex (Materials and Methods; final concentration after mixing, 0.2 μM) with GTP (200 μM) was rapidly mixed in a stopped-flow apparatus with IF3 (2 μM), GTP (200 μM), and EF-G (2 μM) (1) or EF-G (2 μM) and RRF (5 μM) (2). The release of Pi from ribosome-bound EF-G was monitored by the fluorescence increase of MDCC-labeled PBP (2.5 μM) caused by the binding of Pi. The delays are indicated by vertical dotted lines. (B) Pi release in the presence of FA, short time window. Pi release was measured as in A (traces 1 and 2) or in the presence of FA (200 μM) without (trace 3) or with RRF (trace 4). (C) Pi release in the presence of FA, long time window.
