Structural investigation suggests Elongation Factor-G1 is the bona fide canonical translation factor for ribosome recycling in mycobacteria

Bacterial elongation factor G (EF-G) facilitates mRNA and tRNA translocation during peptide elongation as well as promotes splitting of ribosomal subunits during ribosome recycling in coordination with ribosome recycling factor (RRF). Two homologs of EF-G (EF-G1 and EF-G2) have been identified in some bacterial species, including mycobacteria and in mitochondria. We have previously reported that mycobacterial EF-G2 functions as a stress-specific factor. Here, we demonstrate that EF-G1 functions as the canonical elongation factor responsible for ribosome recycling in mycobacteria. In addition, our structural analyses explain why mycobacterial EF-G2 lacks recycling activity, in contrast to its mitochondrial counterpart. We report cryo-EM structures of the M. smegmatis 70S ribosome and 50S subunit in complex with RRF and/or EF-G1 at 3-4 [A] resolution, elucidating the molecular architecture of intermediate structures and mechanistic basis of ribosome recycling. Notably, GTP hydrolysis on EF-G1 is not required for ribosomal disassembly in mycobacteria, unlike in E. coli.