Structural mechanism of GTPase-powered ribosome-tRNA movement

Abstract

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

High-resolution structures of a thermophilic eukaryotic 80S ribosome reveal atomistic details of translocation

“…6c, d ) and thereby activates the catalytic histidine, which flips into the active site to position the catalytic water molecule (not modelled in other cryo-EM structures). GTP hydrolysis, and probably subsequent phosphate release, as shown very recently by detailed time-resolved cryo-EM studies for the bacterial system 61 , 62 , impacts on eEF2-ribosome interactions by modulating and loosening contact sites, as observed in our eEF2-GDP structure between the P-loop and the SRL.…”

“…Upon back-rotation of the 40S subunit, phosphate release is communicated in a relay system via switch 1 to the adjacent domains II and III. Finally eEF2 relaxes, in agreement with the general ‘loaded-spring’ model 63 , as discussed also for EF-G 61 . The back-swiveling of the 40S head as last step of translocation is then able to release eEF2-GDP and to set the stage for a new round of polypeptide elongation.…”

“…Thus, it is not eEF2-GTP that stabilizes the full 80S rotation, but the rotation in situ ‘stabilizes’ eEF2-GTP in the full-activated conformation. How bacterial EF-G is activated by the rotating ribosome is left unexplained here, but recent time-resolved cryo-EM data 61 indicate a different mechanism of sw1 compaction without an arginine finger and including a SRL twisting that modulates sw2 in the transition state of GTP hydrolysis.…”

Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP

“…These findings are similar to those in two cryo-EM studies of ribosomal EF-G complexes with antibiotics published while our manuscript was under review. GDP and Pi were reported in the cryo-EM structure of pretranslocation ribosome stalled with the antibiotic apramycin, which locks the decoding center similarly to viomycin 64 . EF-G-bound ribosome with the antibiotic spectinomycin is also consistent with post-hydrolysis GDP and Pi, although the structural model was reported as GTP 65 (Supplementary Fig.…”

Mechanisms of ribosome recycling in bacteria and mitochondria: a structural perspective

“…Single molecule FRET experiments suggested the existence of a compact EF-G on the ribosome [ 13 ], and a low-resolution cryo-EM reported large domain movements in EF-G on the ribosome [ 14 ]. Despite this, recent time-resolved cryo-EM studies of EF-G bound to the 70S ribosome during tRNA translocation did not observe the compact form of EF-G [ 11 , 12 , 75 ], further suggesting that it is not a ribosome-EF-G state that is highly populated. EF-G in its extended conformation interacts with RRF on the post-termination 70S complex; however, the low-resolution of the available cryo-EM studies limits the interpretation of the specific contacts between EF-G and RRF, and with the ribosome.…”