Fig 6. Biochemical evidence for mRNA release from the PoTC.

PoTC was incubated in buffer R2 at 30°C for 20 min without (A) or with (B) 5 μM of RRF and EF-G and 500 μM GTP. The reaction mixture was subjected to 15–35% SDGC at 47.6 k rpm for 40 min in rotor SW 50.1, and the sedimentation profile was obtained by ISCO. Sedimentation was from left to right. Ribosomal particles and polysome are indicated in the figure. (C) Total tRNA in polysomes or the PoTC (50 pmoles) was released by RRF and EF-G (2.5 μM each) at 30°C for 10 min (squares, and circles, respectively). The released tRNA was aminoacylated with [¹⁴C]-amino acid mixture as described in Materials and Methods. The [¹⁴C]-aminoacyl-tRNA radioactivity was converted to pmoles as described in the supplement. The amount of tRNA thus calculated was plotted against the amount of ribosomal complex (x-axis). (D) The densities of RNA bands (see S3 Fig) were measured and used to calculate the ratio tRNA/5S rRNA and plotted against the incubation period with EF-G and RRF. (E) Sedimentation analysis performed as in Materials and Methods.