Chapter 44 : Mechanism of Bacterial Translation Termination and Ribosome Recycling

Stimulation of RF1 recycling by RF3. Termination complex containing fMet-Phe-Thr-Ile-tRNAIle bound in the P site with a UAA codon in the A site was prepared as described by Freistroffer et al. (1997). (A) The amount of peptidyl-tRNA remaining bound to ribosomes after 2 min of incubation with RF1, with and without 0.9 μM RF3 and with or without 0.2 mM GTP, is shown as a function of the amount of RF1 present. (B) The amount of peptidyl-tRNA remaining bound to ribosomes after incubation with 0.25 pmol of RF1 is shown as in panel A as a function of time of incubation. Stimulation of RF1 recycling is seen to require the presence of GTP. The data are from Freistroffer et al., 1997.

Rapid recycling of ribosomes requires RF3, RRF, and EF-G. Ribosome-recycling times for the translation of a short mRNA encoding fMet-Phe-Leu were measured in vitro as described by Pavlov et al. (1997a) and are shown at the right of each horizontal bar in seconds. For purposes of interpretation, the overall recycling time in the presence of all components is divided arbitrarily into equal periods for initiation, elongation and release, RF release, and the remaining events in ribosome recycling. The horizontal bars interpret the increased recycling time when RRF, RF3, or both factors are omitted in terms of increased times required for RF release and/or ribosome recycling. The two lowest bars show the effect of reducing the concentration of EF-G from 0.8 to 0.3 μM, which increases the overall recycling time in the presence but not in the absence of RRF. The data are from Pavlov et al., 1997a .

RRF restores rapid ribosome recycling inhibited by high concentration of RF1. Ribosome-recycling times for the synthesis of fMet-Phe-Leu were measured in vitro as described by Pavlov et al. (1997b) at three concentrations of RF1 in the absence of RF3. The recycling times are shown as functions of the reciprocal of the RRF concentration present in the translation system. The data, drawn from Pavlov et al., 1997b , show that the increase in recycling time due to RF1 rebinding to the posttermination complex can be countered by increasing the concentration of RRF in the translation system.

RRF, EF-G, and IF3 release deacylated tRNA from the posttermination complex. Termination complexes containing fMet-Phe-Thr-Ile-tRNAIle bound in the P site with a UAA codon in the A site were prepared as described by Karimi et al. (1999) , and the tetrapeptide was released by reaction with puromycin. The dissociation of deacylated tRNAIle was monitored by incubation of the posttermination complexes in the presence of an excess of Ile-tRNA synthetase and other components needed for the rapid aminoacylation of tRNAIle with [14C]Ile, with or without the addition of factors RRF, EF-G, IF3, and RF3 as shown. The data are from Karimi et al., 1999.

RRF and EF-G are required for recycling of the 50S ribosomal subunit, and IF3 is required for recycling of the 30S ribosomal subunit. The synthesis of fMet-Phe-Thr-Ile was measured as described by Karimi et al. (1999) . The translation system contained a constant amount (6 pmol) of active preinitiated 30S subunits and different amounts of active 50S subunits as indicated and as shown by the upper limits of the light-gray (30S) and medium-gray (50S) shaded areas. The experiments were carried out in the absence of RRF and IF3 (○), with 1 μM RRF (△), and with 1 μM (each) of RRF and IF3 (◇). In the absence of RRF and IF3, the amount of tetrapeptide synthesized is limited by the amount of 50S subunit added. In the presence of RRF (and EF-G) but the absence of IF3, the amount of tetrapeptide synthesized is limited by the amount of preinitiated 30S subunits, showing that under these conditions the 50S subunit can recycle. In the presence of both RRF (and EF-G) and IF3, the amount of tetrapeptide synthesized becomes greater than the amount of preinitiated 30S subunits, indicating that both subunits can recycle. The data are from Karimi et al., 1999.

IF3 removes deacylated tRNA from the 30S subunit. 30S ribosomal subunits were programmed with a short mRNA encoding fMet-Phe-Leu and with deacylated tRNAPhe in the P site, as described by Karimi et al. (1999) . The complexes were then incubated in the absence of RRF, EF-G, and IF3 (○), with 1 μM RRF and 1 μM EF-G (△), with 1 μM IF3 (◇), and with 1 μM RRF, 1 μM EF-G, and 1 μM IF3 (□).The dissociation of deacylated tRNAPhe was monitored by charging the tRNA in the presence of an excess of Phe-tRNA synthetase, [14C]Phe, and other components needed for rapid aminoacylation. The data are from Karimi et al., 1999.

Model for the events following peptide release. RF3 catalyzes the dissociation of RF1 (or RF2) from the A site after peptide release ( Freistroffer et al., 1997 ). RRF and EF-G then bind to the 70S posttermination complex and, in a GTPrequiring reaction, provoke the dissociation of the 50S subunit. Finally, IF3 displaces the deacylated tRNA from the 30S posttermination complex ( Karimi et al., 1999 ).

Stimulation of RF2 recycling by RF3 in the presence of different guanine nucleotides. A catalytic amount of RF2 (10 mM) was added to termination complexes (90 nM) containing fMet-Phe-Thr-Ile-tRNAIle bound in the P sites of ribosomes with a UGA(U) stop signal in the A site, and the fraction of the released fMet-Phe-Thr-Ile tetrapeptide was measured as a function of time. RF3 (0.9 μM) and guanine nucleotides (0.2 mM) were added where indicated. The figure shows that the rate of RF2 recycling in the presence of GTP (○) is much faster than with GDPNP (◇) and that RF3 added with GDP (△) is slightly stimulatory. In contrast, RF3 added without guanine nucleotide (□) inhibits RF2 recycling compared to the control in the absence of RF3 (▲).