Structural consequences of the interaction of RbgA with a 50S ribosomal subunit assembly intermediate

Abstract

Bacteria harbor a number GTPases that function in the assembly of the ribosome and are essential for growth. RbgA is one of these GTPases and is required for the assembly of the 50S subunit in most bacteria. Homologs of this protein are also implicated in the assembly of the large subunit of the mitochondrial and eukaryotic ribosome. We present here the cryo-electron microscopy structure of RbgA bound to a Bacillus subtilis 50S subunit assembly intermediate (45SRbgA particle) that accumulates in cells upon RbgA depletion. Binding of RbgA at the P site of the immature particle stabilizes functionally important rRNA helices in the A and P-sites, prior to the completion of the maturation process of the subunit. The structure also reveals the location of the highly conserved N-terminal end of RbgA containing the catalytic residue Histidine 9. The derived model supports a mechanism of GTP hydrolysis, and it shows that upon interaction of RbgA with the 45SRbgA particle, Histidine 9 positions itself near the nucleotide potentially acting as the catalytic residue with minimal rearrangements. This structure represents the first visualization of the conformational changes induced by an assembly factor in a bacterial subunit intermediate.

Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis

“…S9 ). While some of these GTPases have already been identified in bacteria ( 16 , 17 ) ( SI Appendix , Table S3 ), here they were observed acting together on a maturating ribosomal complex at the reported resolution. GTPases have widespread roles in the cell ( 18 ).…”

Structure of the full kinetoplastids mitoribosome and insight on its large subunit maturation

“…5 c and Extended Fig 9). While some of these GTPase have already been identified in bacteria 15,16 (Extended Data Table 3), this is the first time that they are observed acting together on a maturating ribosomal complex at the reported resolution. GTPases have widespread roles in the cell 17 .…”

“…9 a) interacts with H74-75 of domain V and mt-LAF3 that contacts mt-LAF5 through its N-terminal domain. Similarly to the described roles of these proteins in bacteria, they bind the assembly intermediate rRNA to facilitate the folding of domain IV and V helices 15,16 . The third GTPase, mt-LAF4, is homologous to EngB/YihA in bacteria that was structurally characterized alone, outside the context of the maturing LSU.…”

“…9). The bacterial homologs of two of them, EngA (homolog of mt-LAF5) and RbgA (homolog of mt-LAF3) were previously described in context of the 50S 15,16 . Mt-LAF5, in the GTP state, (Extended Data Fig.…”

Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit

“…Studies in yeasts reported that deletion of this protein (Mtg1 in yeast) results in respiration deficiency (Barrientos et al 2003). In B. subtilis, where this assembly factor is essential, the LSU:RbgA complex showed that the Nterminal domain overlaps with rRNA H69 and H71, and that the C-terminal helical domain interacts with H62 and H64 (Seffouh et al 2019). In this position, RbgA displaces the P-site and further interacts with the surrounding rRNA, including H92 and H93.…”