Fig 8. tRNA is released first followed by mRNA release and ribosome splitting during the recycling reaction.

PoTC (0.05 μM) was incubated with RRF (5 μM), EF-G (5 μM) and GTP (500 μM) in the presence of IF3 (4.5 μM) in buffer R1 at 30°C. To stop the reaction at various times, a portion (13 pmol PoTC) of the reaction mixture was taken and placed on ice. The released tRNA (open triangles) from 10 pmol of PoTC was measured as described in Materials and Methods. The rest of the PoTC (3 pmol) was analyzed for mRNA release by ISCO (open circles) and splitting of 70S ribosomes (closed circles). (A) Time course of the disassembly of the PoTC. 100% of reaction, representing complete disassembly, was obtained from exposure of the PoTC to 1 mM Mg²⁺. Inset table show the rate constants of the three reactions (mRNA, tRNA release and splitting of ribosomes). (B) Raw data showing the sedimentation pattern of polysomes and 50S subunits. Sedimentation was from left to right.