SummaryRibosomal RNA (rRNA) contains a number of modified nucleosides in functionally important regions including the intersubunit bridge regions. As the activity of ribosome recycling factor (RRF) in separating the large and the small subunits of the ribosome involves disruption of intersubunit bridges, we investigated the impact of rRNA methylations on ribosome recycling. We show that deficiency of rRNA methylations, especially at positions 1518 and 1519 of 16S rRNA near the interface with the 50S subunit and in the vicinity of the IF3 binding site, adversely affects the efficiency of RRF-mediated ribosome recycling. In addition, we show that a compromise in the RRF activity affords increased initiation with a mutant tRNA fMet wherein the three consecutive G-C base pairs (29GGG31:39CCC41), a highly conserved feature of the initiator tRNAs, were mutated to those found in the elongator tRNA Met (29UCA31:39yGA41). This observation has allowed us to uncover a new role of RRF as a factor that contributes to fidelity of initiator tRNA selection on the ribosome. We discuss these and earlier findings to propose that RRF plays a crucial role during all the steps of protein synthesis.

Specific interactions between ribosome recycling factor (RRF) and elongation factor-G (EFG) mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The interactions between RRF and EFG are also important in peptidyl-tRNA release from stalled pre-termination complexes. Unlike the post-termination complexes (harboring deacylated tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EFG in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, (i) Inclusion of IF3 with RRF and EFG results in recycling of the pre-termination complexes; (ii) IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (iii) Transduction of infC135 (which encodes a functionally compromised IF3) in E.coli LJ14 generates a ‘synthetic severe’ phenotype; (iv) The infC135 and frr1 (containing an insertion in the RRF gene promoter) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E.coli; and (v) IF3 facilitates ribosome recycling by Thermus thermophilus RRF and E.coli EFG in vivo and in vitro. These lines of evidence clearly demonstrate the physiological importance of IF3 in the overall mechanism of ribosome recycling in E.coli.

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Ribosome recycling is a process which dissociates the post-termination complexes (post-TC) consisting of mRNA-bound ribosomes harbouring deacylated tRNA(s). Ribosome recycling factor (RRF), and elongation factor G (EFG) participate in this crucial process to free the ribosomal subunits for a new round of translation. We discuss the over-all pathway of ribosome recycling in eubacteria with especial reference to the important role of the initiation factor 3 (IF3) in this process. Depending on the step(s) at which IF3 function is implicated, three models have been proposed. In model 1, RRF and EFG dissociate the post-TCs into the 50S and 30S subunits, mRNA and tRNA(s). In this model, IF3, which binds to the 30S subunit, merely keeps the dissociated subunits apart by its anti-association activity. In model 2, RRF and EFG separate the 50S subunit from the post-TC. IF3 then dissociates the remaining complex of mRNA, tRNA and the 30S subunit, and keeps the ribosomal subunits apart from each other. However, in model 3, both the genetic and biochemical evidence support a more active role for IF3 even at the step of dissociation of the post-TC by RRF and EFG into the 50S and 30S subunits.

Internal mobility of the two domain molecule of ribosome recycling factor (RRF) is known to be important for its action. Mycobacterium tuberculosis RRF does not complement E. coli for its deficiency of RRF (in the presence of E. coli EF-G alone). Crystal structure had revealed higher rigidity of the M. tuberculosis RRF due to the presence of additional salt bridges between domains. Two inter-domain salt bridges and one between the linker region and the domain containing C-terminal residues were disrupted by appropriate mutations. Except for a C-terminal deletion mutant, all mutants showed RRF activity in E. coli when M. tuberculosis EF-G was also co-expressed. The crystal structures of the point mutants, that of the C-terminal deletion mutant and that of the protein grown in the presence of a detergent, were determined. The increased mobility resulting from the disruption of the salt bridge involving the hinge region allows the appropriate mutant to weakly complement E. coli for its deficiency of RRF even in the absence of simultaneous expression of the mycobacterial EF-G. The loss of activity of the C-terminal deletion mutant appears to be partly due to the rigidification of the molecule consequent to changes in the hinge region.