The Cryo-EM Structure of a Translation Initiation Complex from Escherichia coli

@article{Allen2005TheCS,
  title={The Cryo-EM Structure of a Translation Initiation Complex from Escherichia coli},
  author={Gregory S. Allen and Andrey V Zavialov and Richard P. Gursky and M{\aa}ns Ehrenberg and Joachim Frank},
  journal={Cell},
  year={2005},
  volume={121},
  pages={703-712}
}
The 70S ribosome and its complement of factors required for initiation of translation in E. coli were purified separately and reassembled in vitro with GDPNP, producing a stable initiation complex (IC) stalled after 70S assembly. We have obtained a cryo-EM reconstruction of the IC showing IF2*GDPNP at the intersubunit cleft of the 70S ribosome. IF2*GDPNP contacts the 30S and 50S subunits as well as fMet-tRNA(fMet). IF2 here adopts a conformation radically different from that seen in the recent… 
The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli
TLDR
A cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3, provides insights into the mechanism of mRNA selection during translation initiation.
Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome
TLDR
Two cryo-EM structures of the assembled 70S initiation complex comprising mRNA, fMet-tRNAfMet and IF2 with either a non-hydrolyzable GTP analog or GDP are presented and insights into the molecular mechanism guiding release of IF1 and IF3 are provided.
Structural transitions of translation initiation factor IF2 upon GDPNP and GDP binding in solution.
TLDR
The structural transitions of IF2C upon GDPNP binding and following nucleotide hydrolysis support the concept of cofactor-dependent conformational switching rather than the classical model for GTPase activity.
Interactions of the release factor RF1 with the ribosome as revealed by cryo-EM.
TLDR
Cryo-electron microscopy results obtained in this study show that ribosome-bound RF1 is in an open conformation, unlike the closed conformation observed in the crystal structure of the free factor, allowing its simultaneous access to both the decoding center and the peptidyl-transferase center.
Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor
TLDR
Fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining and the role played by the Nterminus of IF2 in this process.
Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association
TLDR
The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation.
Kinetic Dissection of Translation Initiation in Prokaryotes.
TLDR
A potential checkpoint for mRNA selection during translation initiation is identified on the basis of kinetic constants determined in the current work, which will provide important information about the conformation and contacts of IF2 with the ribosome during the dynamic process of translation initiation.
Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1
TLDR
The cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1 is presented and provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3.
Interaction of the G' domain of elongation factor G and the C-terminal domain of ribosomal protein L7/L12 during translocation as revealed by cryo-EM.
TLDR
Molecular analyses of the cryo-EM maps show that three structural components participate in formation of the ALC, and that both EF-G and the ribosomal protein L7/L12 undergo large conformational changes to form the A LC.
Structural insight into the recognition of amino-acylated initiator tRNA by eIF5B in the 80S initiation complex
TLDR
A reinterpretation of the 6.6 Å resolution cryo-electron microscopy structure of the eukaryal 80S initiation complex using the recently published crystal structure of eIF5B reveals that domain IV of e IF5B forms extensive interaction interfaces with the Met-tRNAi, which directly involve the methionylated 3′ CCA-end of the acceptor stem.
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References

SHOWING 1-10 OF 78 REFERENCES
Ribosomal localization of translation initiation factor IF2.
TLDR
A model for the positioning of IF2 in the 70S initiation complex as determined by cleavage of rRNA by the chemical nucleases Cu(II):1,10-orthophenanthroline and Fe(II:EDTA tethered to cysteine residues introduced into IF2 is presented.
Characterization of the domains of E. coli initiation factor IF2 responsible for recognition of the ribosome
TLDR
It is concluded that the N‐terminus of IF2 is required for optimal interaction of the factor with both 30S and 50S ribosomal subunits, and a structural model for the interaction of IF1 with the ribosome is presented.
Crystal structure of an initiation factor bound to the 30S ribosomal subunit.
TLDR
The crystal structure of a complex of IF1 and the 30S ribosomal subunit is reported, explaining how localized changes at the ribosome A site lead to global alterations in the conformation of the30S subunit.
Binding of Escherichia coli initiation factor IF2 to 30S ribosomal subunits: a functional role for the N-terminus of the factor.
TLDR
It is concluded that the N-terminus of IF2 has affinity per se to bind the ribosomal subunit, with domain II being directly involved in the interaction.
Location of translational initiation factor IF3 on the small ribosomal subunit.
TLDR
The placement of IF3 on the 30S subunit of the Thermus thermophilus ribosome allows an understanding in structural terms of the biochemical functions of this initiation factor, namely its ability to dissociate 70S ribosomes into 30S and 50S subunits and the preferential selection of initiator tRNA by IF3 during initiation.
Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy
TLDR
A cryo-electron microscopy study presents a mechanism that can explain the sequence of events during the decoding of mRNA on the ribosome, and proposes a mechanism to facilitate codon recognition by the incoming aa-tRNA and to provide the codon-anticodon recognition-dependent signal for the GTPase activity of EF-Tu.
X-Ray Structures of the Universal Translation Initiation Factor IF2/eIF5B Conformational Changes on GDP and GTP Binding
TLDR
X-ray structures of the universal translation initiation factor IF2/eIF5B have been determined and a molecular lever is revealed, which amplifies a modest conformational change in the Switch 2 region of the G domain induced by Mg(2+)/GTP binding over a distance of 90 A from the G kingdom center to domain IV.
A ratchet-like inter-subunit reorganization of the ribosome during translocation
TLDR
Three-dimensional cryo-electron microscopy maps of the Escherichia coli 70S ribosome in various functional states show that both EF-G binding and subsequent GTP hydrolysis lead to ratchet-like rotations of the small 30S sub unit relative to the large 50S subunit, indicating a two-step mechanism of translocation.
The solution structure of the Escherichia coli initiator tRNA and its interactions with initiation factor 2 and the ribosomal 30 S subunit.
TLDR
A three-dimensional model of the initiator tRNA is presented, which displays several differences with yeast tRNAPhe, and additional protections are observed in the acceptor stem and in the anticodon arm, resulting from a strong steric hindrance and from the codon-anticodon interaction within the subunit decoding site.
A study of the interaction of Escherichia coli initiation factor IF2 with formylmethionyl‐tRNAMet f by partial digestion with cobra venom ribonuclease
TLDR
It is shown that IF-2 specifically protected the formylated form of the initiator tRNA, ~dicating thatIF-2 interacts with the amino acid acceptor region of the Initiator t RNA, independent of GTP.
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