Abstract:During translation, a conserved GTPase elongation factor—EF-G in bacteria or eEF2 in eukaryotes—translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of … Show more
“…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.…”
Section: Resultssupporting
confidence: 77%
“…Furthermore, comparing our and other (TI)-POST structures including the complete tRNA 2 •mRNA module 10 , allow for the assignment of the Dph conformation to a defined post-state (a Dph post - pawl position) upon GTP hydrolysis. The post-state conformation of domain IV corresponds to the time-resolved cryo-EM data obtained very recently for EF-G 62 , however, Dph is not present in bacteria. Fig.…”
Ribosomes are complex and highly conserved ribonucleoprotein assemblies catalyzing protein biosynthesis in every organism. Here we present high-resolution cryo-EM structures of the 80S ribosome from a thermophilic fungus in two rotational states, which due to increased 80S stability provide a number of mechanistic details of eukaryotic translation. We identify a universally conserved ‘nested base-triple knot’ in the 26S rRNA at the polypeptide tunnel exit with a bulged-out nucleotide that likely serves as an adaptable element for nascent chain containment and handover. We visualize the structure and dynamics of the ribosome protective factor Stm1 upon ribosomal 40S head swiveling. We describe the structural impact of a unique and essential m1acp3 Ψ 18S rRNA hyper-modification embracing the anticodon wobble-position for eukaryotic tRNA and mRNA translocation. We complete the eEF2-GTPase switch cycle describing the GDP-bound post-hydrolysis state. Taken together, our data and their integration into the structural landscape of 80S ribosomes furthers our understanding of protein biogenesis.
“…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.…”
Section: Resultssupporting
confidence: 77%
“…Furthermore, comparing our and other (TI)-POST structures including the complete tRNA 2 •mRNA module 10 , allow for the assignment of the Dph conformation to a defined post-state (a Dph post - pawl position) upon GTP hydrolysis. The post-state conformation of domain IV corresponds to the time-resolved cryo-EM data obtained very recently for EF-G 62 , however, Dph is not present in bacteria. Fig.…”
Ribosomes are complex and highly conserved ribonucleoprotein assemblies catalyzing protein biosynthesis in every organism. Here we present high-resolution cryo-EM structures of the 80S ribosome from a thermophilic fungus in two rotational states, which due to increased 80S stability provide a number of mechanistic details of eukaryotic translation. We identify a universally conserved ‘nested base-triple knot’ in the 26S rRNA at the polypeptide tunnel exit with a bulged-out nucleotide that likely serves as an adaptable element for nascent chain containment and handover. We visualize the structure and dynamics of the ribosome protective factor Stm1 upon ribosomal 40S head swiveling. We describe the structural impact of a unique and essential m1acp3 Ψ 18S rRNA hyper-modification embracing the anticodon wobble-position for eukaryotic tRNA and mRNA translocation. We complete the eEF2-GTPase switch cycle describing the GDP-bound post-hydrolysis state. Taken together, our data and their integration into the structural landscape of 80S ribosomes furthers our understanding of protein biogenesis.
“…These observations provide insights into how IF2-GDP dissociates from the ribosome. As the 30 S subunit rotates back, compact IF2 releases its hold from the ribosome in a stepwise fashion; the G-domain peels off from the SRL and domain II dissociates from the ribosome before the other domains C1 and C2, similar to the proposed dissociation mechanism of EF-Tu and EF-G from the elongating ribosome 28 , 29 . Fig.…”
During translation initiation, initiation factor 2 (IF2) holds initiator transfer RNA (fMet-tRNAifMet) in a specific orientation in the peptidyl (P) site of the ribosome. Upon subunit joining IF2 hydrolyzes GTP and, concomitant with inorganic phosphate (Pi) release, changes conformation facilitating fMet-tRNAifMet accommodation into the P site and transition of the 70 S ribosome initiation complex (70S-IC) to an elongation-competent ribosome. The mechanism by which IF2 separates from initiator tRNA at the end of translation initiation remains elusive. Here, we report cryo-electron microscopy (cryo-EM) structures of the 70S-IC from Pseudomonas aeruginosa bound to compact IF2-GDP and initiator tRNA. Relative to GTP-bound IF2, rotation of the switch 2 α-helix in the G-domain bound to GDP unlocks a cascade of large-domain movements in IF2 that propagate to the distal tRNA-binding domain C2. The C2-domain relocates 35 angstroms away from tRNA, explaining how IF2 makes way for fMet-tRNAifMet accommodation into the P site. Our findings provide the basis by which IF2 gates the ribosome to the elongation phase.
“…In the rotated/hybrid state, the SSU rotates with respect to the LSU and tRNAs move into hybrid (A/P, A/P*, and P/E) conformations, in which the tRNA anticodons remain in the A and P site, respectively, while the acceptor arms move towards the P and E site on the LSU 19 – 21 , 23 – 37 . The elbow region of the A/P-site tRNA is mobile and can adopt slightly different orientations, A/P and A/P* 19 , 24 , 25 . EF-G binding facilitates the movement of tRNAs into the hybrid states, as well as rotation of the ribosomal subunits relative to each other 21 , 23 , 30 , 38 – 42 .…”
When reading consecutive mRNA codons, ribosomes move by exactly one triplet at a time to synthesize a correct protein. Some mRNA tracks, called slippery sequences, are prone to ribosomal frameshifting, because the same tRNA can read both 0- and –1-frame codon. Using smFRET we show that during EF-G-catalyzed translocation on slippery sequences a fraction of ribosomes spontaneously switches from rapid, accurate translation to a slow, frameshifting-prone translocation mode where the movements of peptidyl- and deacylated tRNA become uncoupled. While deacylated tRNA translocates rapidly, pept-tRNA continues to fluctuate between chimeric and posttranslocation states, which slows down the re-locking of the small ribosomal subunit head domain. After rapid release of deacylated tRNA, pept-tRNA gains unconstrained access to the –1-frame triplet, resulting in slippage followed by recruitment of the –1-frame aa-tRNA into the A site. Our data show how altered choreography of tRNA and ribosome movements reduces the translation fidelity of ribosomes translocating in a slow mode.
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