Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

Abstract

Ribosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

Types and Functions of Mitoribosome-Specific Ribosomal Proteins across Eukaryotes

“…This is also the case for all the maturation factors involved in mitoribosome biogenesis. Recently, several studies managed to capture mitoribosome assembly intermediates from humans [ 127 , 128 , 129 , 130 , 131 ] and kinetoplastids [ 95 , 98 , 99 , 132 , 133 ], describing different steps and factors involved in the maturation of the large and small mitoribosomal subunits. A portion of these factors were previously described by non-structural means [ 59 , 134 , 135 , 136 ], but most of them were described for the first time with these studies, and most importantly, directly observed in action.…”

Maintaining mitochondrial ribosome function: The role of ribosome rescue and recycling factors

“…Recently, the GTP-binding protein GTPBP6, a human homolog of bacterial HflX, was identified as an essential mitochondrial protein with dual functions [ 132 ]. In addition to its role in mitoribosome biogenesis, GTPBP6 acts as an alternative recycling factor in human mitochondria [ 132 , 133 ]. Similar to the canonical and HflX-driven recycling systems in bacteria, GTPBP6 binding to the 55S mitoribosome facilitates disruption of the critical B2a intersubunit bridge by helix 69 dislocation.…”

“…However, GTPBP6 utilizes a different mechanism to dissociate mitoribosomes. Cryo-EM analysis of post-splitting mtLSU complexes revealed that GTPBP6 abstracts helix 69 by insertion of the Trp107 residue [ 133 ]. Interestingly, kinetic measurements suggest that GTPBP6-mediated ribosome splitting occurs more efficiently on vacant ribosomes or on PoTC [ 132 ].…”

“…Interestingly, kinetic measurements suggest that GTPBP6-mediated ribosome splitting occurs more efficiently on vacant ribosomes or on PoTC [ 132 ]. Further structural analysis revealed that GTPBP6 binding to the 55S mitoribosome requires the deacylated tRNA in the P site as the presence of a polypeptide would sterically exclude GTPBP6 association [ 133 ]. Apparently, accommodation of GTPBP6, similar to mtRRF, favours the rotated state of the mitoribosome, which is achievable only with an uncharged P site tRNA [ 45–47 ].…”

Mitoribosomal small subunit maturation involves formation of initiation-like complexes

“…Recently, first mechanistic insights into the assembly of mitoribosomes were obtained by structure determinations of the kinetoplastid small (mt-SSU) ( 31 , 32 ) and large subunit (mt-LSU) ( 32 – 34 ) as well as late-stage human mt-LSU assembly intermediates ( 35 – 40 ). The study of untagged Trypanosoma brucei mt-SSU resulted in several cryogenic electron microscopy (cryo-EM) structures of assembly intermediates that illustrated possible assembly progression.…”