Interaction of RRF and EF-G from E. coli and T. thermophilus with ribosomes from both origins—insight into the mechanism of the ribosome recycling step

Abstract

Ribosome recycling factor (RRF), elongation factor-G (EF-G), and ribosomes from Thermus thermophilus (tt-) and Escherichia coli (ec-) were used to study the disassembly mechanism of post-termination ribosomal complexes by these factors. With tt-RRF, ec-EF-G can release bound-tRNA from ec-model post-termination complexes. However, tt-RRF is not released by ec-EF-G from ec-ribosomes. This complex with tt-RRF and ec-ribosomes after the tRNA release by ec-EF-G is regarded as an intermediate of the disassembly reaction. Not only tt-RRF, but also mRNA, cannot be released from ec-ribosomes by tt-RRF and ec-EF-G. These data suggest that the release of RRF from ribosomes is coupled or closely related to the release of mRNA during disassembly of post-termination complexes. With tt-ribosomes, ec-EF-G cannot release ribosome-bound ec-RRF even though they are from the same species, showing that proper interaction of ec-RRF and ec-EF-G does not occur on tt-ribosomes. On the other hand, in contrast to a published report, tt-EF-G functions with ec-RRF to disassemble ec-post-termination complexes. In support of this finding, tt-EF-G translocates peptidyl tRNA on ec-ribosomes and catalyzes ec-ribosome-dependent GTPase, showing that tt-EF-G has in vitro translocation activity with ec-ribosomes. Since tt-EF-G with ec-RRF can release tRNA from ec-post-termination complexes, the data are consistent with the hypothesis that the release of tRNA by RRF and EF-G from post-termination complexes is a result of a translocation-like activity of EF-G on RRF.

The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits

“…In the first step, deacylated tRNA is released from the complex A by RRF and EF-G (Hirashima and Kaji 1973;Hirokawa et al 2002), resulting in the intermediate complex, complex B. Recent experiments suggested that RRF moves to the second site on the ribosome during this process from A to B in a fashion analogous to that of tRNA movement during the translocation step (Hirokawa et al 2002;Kiel et al 2003;Raj et al 2005). The next step is still uncertain.…”

“…Otherwise, two RRF molecules (one for A through B to C and the other for C to C 0 ) would be required for the recycling of one ribosome, because the release of the initially bound RRF from the ribosome is necessary to release mRNA (Raj et al 2005). The dissociation of 70S ribosomes into subunits and the release of ribosomes from mRNA by RRF and EF-G measured by SDGC appear to share the same mechanism, as suggested by their equal sensitivity to the EF-G inhibitors (Table 4; Fig.…”

“…Domain I of RRF is known to interact with helices 69 and 71 of 23S rRNA (Lancaster et al 2002;Agrawal et al 2004;Wilson et al 2004). Since these helices 69 and 71 of 23S rRNA interact with helix 44 of 16S rRNA forming the intersubunit bridges B2a and B3, respectively (Yusupov et al 2001), the possible breakage of bridges B2a and B3 by the EF-G-dependent movement of RRF (Hirokawa et al 2002;Kiel et al 2003;Raj et al 2005) may cause the transient dissociation into subunits. However, we believe that the possible disruption of B2a and B3 by RRF is not enough to cause the dissociation of ribosomes into subunits.…”

The ribosome-recycling step: consensus or controversy?

“…Regarding the splitting of the 70S ribosome, three laboratories support this reaction [16, 17 and 18]. Evidence for the order of the first two steps comes from the fact that the intermediate complex (mRNA-70S ribosome complex without tRNA) can be isolated in the presence of low concentrations of inhibitor [21] or with the use of Thermus thermophilus RRF [38]. However, this does not preclude the possibility that the first two steps might occur simultaneously.…”

“…A recent report has shown that Thermus thermophilus RRF, which does not function in Escherichia coli in vivo [36], can complement the defective E. coli RRF if a plasmid expressing IF3 is present [37]. Because T. thermophilus RRF, together with E. coli EF-G and ribosomes, functions only to release tRNA [38], an excess of IF3 might disassemble the 70S ribosomes left on mRNA without tRNA. These data suggest the possibility that excess IF3 might participate actively in the splitting of 70S ribosomes in vivo.…”