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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

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India¹

Author for correspondence: Umesh Varshney. Tel: +91 80 394 2686. Fax: +91 80 360 2697 or 0683. e-mail: varshney{at}mcbl.iisc.ernet.in

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 cross-species-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.

Keywords: tubercle bacilli, Thermus thermophilus, elongation factor G, polysome binding, termination complex

Abbreviations: CD, circular dichroism; EcoEFG, E. coli EFG; EcoRRF, full-length E. coli RRF; EFG, elongation factor G; frr, locus coding for RRF; frr^ts, locus coding for temperature-sensitive RRF; ESI-MS, electron spray ionization-mass spectroscopy; MtuEFG, M. tuberculosis EFG; MtuRRF, full-length M. tuberculosis RRF; RFF, ribosome recycling factor; TthRRF, full-length T. thermophilus RRF

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