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

Allen, Gregory S.; Zavialov, Andrey V.; Gursky, Richard; Ehrenberg, Måns; Frank, Joachim

doi:10.1016/j.cell.2005.03.023

Cited by 261 publications

(318 citation statements)

References 64 publications

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“…The foot-printing studies were done with 30S subunits and not tried with 70S ribosomes. As mentioned in the Introduction, evidence for IF3 presence on 70S ribosomes was reported (9). The overlapping binding sites on 30S and 70S (9) could be reconciled with a distinct binding region, whereas the binding site derived from a crystallographic study could not, because the C-terminal domain of IF3 was assigned to the upper end of the shoulder on the solvent side of 30S subunits of Thermus thermophilus (51).…”

Section: Discussionmentioning

confidence: 84%

“…However, the in vivo concentration of IF3 is 100-fold less (8) than required for full dissociation of 70S in vitro (4). Evidence for the presence of IF3 on 70S ribosomes was reported (9), indicating that the functional spectrum of IF3 is possibly not restricted to an antiassociation effect. Both IF3 and IF2 are also responsible for the fidelity of decoding the initiation AUG by fMet-tRNA Met f at the P site of 30S subunits (10).…”

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confidence: 99%

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70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Yamamoto

Wittek

Gupta

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

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According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1-IF3). Here, we describe a novel type of initiation termed "70S-scanning initiation," where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine-Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.protein synthesis | ribosomal functions | translational initiation | 30S-binding initiation | 70S-scanning initiation I t is textbook knowledge that 30S subunits initiate protein synthesis in bacteria; they recognize the initiation site of the mRNA composed of the Shine-Dalgarno (SD) sequence, the AUG codon, and fMet-tRNA, together with three initiation factors (IFs) forming the 30S initiation complex (30SIC). Association of the large 50S subunit triggers the release of the IFs, leading to the 70S initiation complex (70SIC) that enters the elongation phase of translation (reviewed in 1). We term this initiation path the "30S-binding mode" of bacterial initiation. After elongation and termination, it is thought that the ribosome dissociates into its subunits, thus providing 30S subunits for the next round of initiation.The functional role of IF2 is well defined. It can bind directly to the 30S, providing a docking site for fMet-tRNA (2), but it can also enter the 30S subunit as ternary complex fMet-tRNA•IF2•GTP (3). Both IF2 and IF3 are essential for viability. IF3 has a binding site at the 30S interface (4), which explains its antiassociation effect (5, 6), as well as its role in dissociation of the terminating 70S ribosome (7). However, the in vivo concentration of IF3 is 100-fold less (8) than required for full dissociation of 70S in vitro (4). Evidence for the presence of IF3 on 70S ribosomes was reported (9), indicating that the functional spectrum of IF3 is possibly not restricted to an antiassociation effect. Both IF3 and IF2 are also responsible for the fidelity of decoding the initiation AUG by fMet-tRNA Met f at the P site of 30S subun...

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Section: Discussionmentioning

confidence: 84%

mentioning

confidence: 99%

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Yamamoto

Wittek

Gupta

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

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“…11,14 Thus, a majority of structural data connect uL11 with the elongation cycle of protein synthesis. However, several structural analyses implicate this protein in interaction with IF2 and RF3, indicating involvement of uL11 in the initiation and termination steps of translation, 15,16 which is also supported by several functional analyses. 17,18 The large ribosomal subunit depleted of this protein is able to bind initiation factor IF2 in vitro but it cannot stimulate GTP hydrolysis required for the subunit joining.…”

Section: Introductionmentioning

confidence: 85%

Functional analysis of the uL11 protein impact on translational machinery

Wawiórka¹,

Molestak²,

Szajwaj³

et al. 2016

Cell Cycle

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The ribosomal GTPase associated center constitutes the ribosomal area, which is the landing platform for translational GTPases and stimulates their hydrolytic activity. The ribosomal stalk represents a landmark structure in this center, and in eukaryotes is composed of uL11, uL10 and P1/P2 proteins. The modus operandi of the uL11 protein has not been exhaustively studied in vivo neither in prokaryotic nor in eukaryotic cells. Using a yeast model, we have brought functional insight into the translational apparatus deprived of uL11, filling the gap between structural and biochemical studies. We show that the uL11 is an important element in various aspects of 'ribosomal life'. uL11 is involved in 'birth' (biogenesis and initiation), by taking part in Tif6 release and contributing to ribosomal subunit-joining at the initiation step of translation. uL11 is particularly engaged in the 'active life' of the ribosome, in elongation, being responsible for the interplay with eEF1A and fidelity of translation and contributing to a lesser extent to eEF2-dependent translocation. Our results define the uL11 protein as a critical GAC element universally involved in trGTPase 'productive state' stabilization, being primarily a part of the ribosomal element allosterically contributing to the fidelity of the decoding event.

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“…This process is undertaken by eIF5B, a partial homologue of eubacterial IF2, but more closely related to archaeal aIF5B. The structural details of how eIF5B could concurrently check for both the presence of tRNAi and the sarcin-ricin loop in the context of the large ribosomal subunit had only previously been established at low resolution for the partial bacterial homologue, IF2, which functions in a somewhat different manner [22,23]. Two key cryo-EM structures [24,25], in conjunction with an important crystallographic study of the ribosome [26], have revealed the mechanism by which this takes place.…”

Section: Incorporating the Large Subunit: Coming Full Circlementioning

confidence: 99%

Eukaryotic aspects of translation initiation brought into focus

Aylett

Ban

2017

Phil. Trans. R. Soc. B

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One contribution of 11 to a theme issue 'Perspectives on the ribosome'. In all organisms, mRNA-directed protein synthesis is catalysed by ribosomes. Although the basic aspects of translation are preserved in all kingdoms of life, important differences are found in the process of translation initiation, which is rate-limiting and the most important step for translation regulation. While great strides had been taken towards a complete structural understanding of the initiation of translation in eubacteria, our understanding of the eukaryotic process, which includes numerous eukaryotic-specific initiation factors, was until recently limited owing to a lack of structural information. In this review, we discuss recent results in the field that provide an increasingly complete molecular description of the eukaryotic initiation process. The structural snapshots obtained using a range of methods now provide insights into the architecture of the initiation complex, start-codon recognition by the initiator tRNA and the process of subunit joining. Future advances will require both higher-resolution insights into previously characterized complexes and mapping of initiation factors that control translation on an additional level by interacting only peripherally or transiently with ribosomal subunits.This article is part of the themed issue 'Perspectives on the ribosome'. Eubacterial translation initiationAt its most basic level, the process of translation initiation encompasses only a few key steps. The mRNA molecule undergoing translation must bind to the mRNA channel on the small ribosomal subunit such that the start-codon is positioned into the P-( peptidyl) decoding site. A charged initiator tRNA, in prokaryotes generally (formyl) methionyl-tRNAi, must then be bound at the aforementioned start-codon, decoding the first triplet of the encoded protein establishing the frame in which the eventual addition of the following tRNA adaptor molecules will be incorporated. Finally, the large ribosomal subunit joins the small subunit to form ribosomal complex with initiator tRNA in the P site of the ribosome primed for the elongation stage of protein synthesis during which protein is synthesized based on the message encoded in the RNA. Owing to the large investment of energy involved in translation, this entire process is tightly regulated at the stage of initiation, reducing the wasteful expenditure of resources. In eubacteria, translation initiation is facilitated by two universally conserved factors, IFs (initiation factors) 1 (note that this homologue of eubacterial IF1 in eukaryotes is named eIF1A: eIF1 is a separate factor) and 2, and a region of rRNA dubbed the anti-Shine-Dalgarno sequence [1,2]. The position of the first codon within the decoding site is established by base pairing between the Shine-Dalgarno sequence, located upstream of the first codon within the mRNA, and its complement within the ribosomal rRNA [2]. The separation between the two mRNA elements then defines the start site for translation [2]. Recruitment ...

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

Cited by 261 publications

References 64 publications

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Functional analysis of the uL11 protein impact on translational machinery

Eukaryotic aspects of translation initiation brought into focus

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