Characterization of Mycobacterium tuberculosis ribosome recycling factor (RRF) and a mutant lacking six amino acids from the C-terminal end reveals that the C-terminal residues are important for its occupancy on the ribosome

Abstract

Ribosome recycling factor (RRF), coded for by the frr locus, is involved in the disassembly of post-termination complexes and recycling of the ribosomes for a fresh round of initiation in bacteria and in eukaryotic organelles. In a crossspecies-complementation experiment, it was shown that the Thermus thermophilus RRF protein lacking five amino acids from its C-terminal end (∆C5TthRRF) but not the full-length protein (TthRRF) complemented Escherichia coli for its frr ts phenotype. It was also shown that the Mycobacterium tuberculosis RFF protein (MtuRRF) did not complement E. coli LJ14 for frr ts . However, simultaneous expression of elongation factor G (EFG) and RRF from M. tuberculosis resulted in complementation of E. coli LJ14. Here it is shown that unlike ∆C5TthRRF, an equivalent mutant of MtuRRF lacking six amino acids from its C-terminal end (∆C6MtuRRF) did not complement E. coli LJ14. Surprisingly, ∆C6MtuRRF failed to complement the strain even in the presence of homologous EFG (MtuEFG). The biochemical and biophysical characterization of these proteins suggested that the mutant RRF folded properly. However, ribosome-binding assays showed that the mutant protein was compromised in its binding to E. coli ribosomes. It is suggested that the conserved amino acids at the C-terminal end of the RRFs contribute to their residency on ribosomes and that the specific interactions between RRF and EFG are crucial in the disassembly of the termination complex.

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

“…Again, the rRNA residues in the close neighborhoods of these contacts are modified bases, methyl-U1939 and methyl-C1962. Observation of an interaction of RRF, involving its C terminus, is consistent with genetic studies (31,32) showing that this region of RRF is important for its occupancy on the ribosome.…”

X-ray Structural Studies of Mycobacterium tuberculosis RRF and a Comparative Study of RRFs of Known Structure. Molecular Plasticity and Biological Implications

“…11,18 Significant studies have been carried out on MtRRF, the RRF from the pathogen Mycobacterium tuberculosis. 2,19 Interestingly, these studies have demonstrated MtRRF to possess certain properties different from the two widely studied RRFs, EcRRF and TtRRF. Cross-species complementation experiments show that MtRRF completely fails to function in E. coli, 2 while TtRRF shows residual function.…”

Recycling of the Posttermination Complexes of Mycobacterium smegmatis and Escherichia coli Ribosomes Using Heterologous Factors

“…30 MsmRRF, MtuIF3, and His 6 -MtuEFG from pET14bMtuEFG, and MsmEFG from pRSETBMsmEFG were purified as described in Supplementary Material.…”