The Cryo-EM Structure of a Translation Initiation Complex from <i>Escherichia coli</i>

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

doi:10.1142/9789813234864_0037

Cited by 16 publications

(57 citation statements)

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“…A key structural finding that is consistent between EF‐G, RF3, and IF2 is that each GTPase binds 70S ribosomes in a counterclockwise rotation , which is concomitant with direct connections to the L12 CTD. In contrast to these structural observations, LepA‐bound ribosomes have been reported to be in either an unrotated or clockwise‐rotated 70S conformation, and no connection between LepA and L7/L12 has been observed .…”

Section: Discussionmentioning

confidence: 82%

Ribosomal protein L7/L12 is required for GTPase translation factors EF‐G, RF3, and IF2 to bind in their GTP state to 70S ribosomes

et al. 2017

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Ribosomal protein L7/L12 is associated with translation initiation, elongation and termination by the 70S ribosome. The GTPase activity of EF-G requires the presence of L7/L12, which is critical for ribosomal translocation. Here, we have developed new methods for the complete depletion of L7/L12 from E. coli 70S ribosomes to analyze the effect of L7/L12 on the activities of the GTPase factors EF-G, RF3, IF2 and LepA. Upon removal of L7/L12 from ribosomes, the GTPase activities of EF-G, RF3 and IF2 decreased to basal levels while the activity of LepA decreased marginally. Upon reconstitution of ribosomes with recombinant L12, the GTPase activities of all GTPases returned to full activity. Moreover, ribosome binding assays indicated that EF-G, RF3 and IF2 require L7/L12 for stable binding in the GTP state, and LepA retained >50% binding. Lastly, an EF-GΔG′ truncation mutant possessed ribosome-dependent GTPase activity, which was insensitive to L7/L12. Our results indicate that L7/L12 is required for stable binding of ribosome-dependent GTPases that harbor direct interactions to the L7/L12 CTD, either through a G′ domain (EF-G, RF3) or a unique NTD (IF2). Further, we hypothesize this interaction is concomitant with counter-clockwise ribosomal intersubunit rotation, which is required for translocation, initiation and post-termination.

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Section: Discussionmentioning

confidence: 82%

Ribosomal protein L7/L12 is required for GTPase translation factors EF‐G, RF3, and IF2 to bind in their GTP state to 70S ribosomes

et al. 2017

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“…Rapid and accurate 70S IC formation is promoted by 30S IC-bound initiation factor (IF) 1 and the guanosine triphosphatase (GTPase) IF2, both of which must ultimately dissociate from the 70S IC before the resulting 70S EC can begin translation elongation 1 . Although comparison of 30S 2–6 and 70S 5,7–9 IC structures have revealed that the ribosome, IFs, and fMet-tRNA fMet can acquire different conformations in these complexes, the timing of conformational changes during 70S IC formation, structures of any intermediates formed during these rearrangements, and contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, lack of an authentic 70S EC structure has precluded an understanding of ribosome, IF, and fMet-tRNA fMet rearrangements that occur upon maturation of a 70S IC into a 70S EC.…”

mentioning

confidence: 99%

Real-time structural dynamics of late steps in bacterial translation initiation visualized using time-resolved cryogenic electron microscopy

Kaledhonkar

Zhang

Caban

et al. 2018

Preprint

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Bacterial translation initiation entails the tightly regulated joining of the 50S23 ribosomal subunit to an initiator transfer RNA (fMet-tRNA fMet )-containing 30S ribosomal 24 initiation complex (IC) to form a 70S IC that subsequently matures into a 70S elongation-25 2 competent complex (70S EC). Rapid and accurate 70S IC formation is promoted by 30S IC-26 bound initiation factor (IF) 1 and the guanosine triphosphatase (GTPase) IF2, both of which 27 must ultimately dissociate from the 70S IC before the resulting 70S EC can begin 28 translation elongation 1 . Although comparison of 30S 2-6 and 70S 5,7-9 IC structures have 29 revealed that the ribosome, IFs, and fMet-tRNA fMet can acquire different conformations in 30 these complexes, the timing of conformational changes during 70S IC formation, 31 structures of any intermediates formed during these rearrangements, and contributions 32 that these dynamics might make to the mechanism and regulation of initiation remain 33 unknown. Moreover, lack of an authentic 70S EC structure has precluded an 34 understanding of ribosome, IF, and fMet-tRNA fMet rearrangements that occur upon 35 maturation of a 70S IC into a 70S EC. Using time-resolved cryogenic electron microscopy 36 (TR cryo-EM) 10 we report the first, near-atomic-resolution view of how a time-ordered 37 series of conformational changes drive and regulate subunit joining, IF dissociation, and 38 fMet-tRNA fMet positioning during 70S EC formation. We have found that, within ~20-80 ms, 39 rearrangements of the 30S subunit and IF2, uniquely captured in its GDP•Pi-bound state, 40 stabilize fMet-tRNA fMet in its intermediate, '70S P/I', configuration 7 and trigger dissociation 41 of IF1 from the 70S IC. Within the next several hundreds of ms, dissociation of IF2 from the 42 70S IC is coupled to further remodeling of the ribosome that positions fMet-tRNA fMet into 43 its final, 'P/P', configuration within the 70S EC. Our results demonstrate the power of TR 44 cryo-EM to determine how a time-ordered series of conformational changes contribute to 45

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“…Gst IF2‐G3 is structurally homologous to other OB‐fold proteins, including ribosomal protein S10, aspartyl tRNA synthetase, the C2‐subdomain of Gst IF2, and cold shock protein CspA. A common property to many of these proteins is their capacity to bind RNA . Results obtained for hydroxyl radical cleavage by Fe(II)‐EDTA tethered to cysteine residues in IF2, for amino acid substitutions (Gly420 and Glu424), for cryo‐EM reconstructions, as well as for titrations with ribosomal subunits (RD, unpublished results) indicate that IF2‐G3 is indeed involved in binding of 30S ribosomal subunits and participates in joining the small and large ribosomal subunits during the transition from 30S IC to 70S IC .…”

Section: Resultsmentioning

confidence: 99%

“…G2 contains a guanosine nucleotide binding motif and is able to bind 50S ribosomal subunits, albeit with low affinity . It has been suggested that G3 facilitates the interaction of IF2 with the 30S and possibly also with the 50S ribosomal subunit . C‐terminally from G3 are subdomains C1 and C2.…”

Section: Introductionmentioning

confidence: 99%

“…29 It has been suggested that G3 facilitates the interaction of IF2 with the 30S and possibly also with the 50S ribosomal subunit. 19,[22][23][24][25] C-terminally from G3 are subdomains C1 and C2. The likely function of C1 is to establish contacts with G2/G3 and to mediate communication between these G subdomains and C2, the most C-terminal subdomain of IF2 that was shown to be necessary and sufficient to recognize and bind fMet-tRNA.…”

Section: Introductionmentioning

confidence: 99%

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A model for the interaction of the G3‐subdomain of Geobacillus stearothermophilus IF2 with the 30S ribosomal subunit

et al. 2016

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Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet-tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2-30S interaction, is positioned between the GTP-binding G2 and the fMet-tRNA binding Cterminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two b-sheets with each four anti-parallel strands, followed by a C-terminal a-helix. In line with its role as linker between G3 and subdomain C1, this helix has no well-defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB-fold module, similar to that of the corresponding subdomain of Thermus thermophilus IF2, and to that of other known RNAbinding modules such as IF2-C2, IF1 and subdomains II of elongation factors EF-Tu and EF-G. Structural comparisons have resulted in a model that describes the interaction between IF2-G3 and the 30S ribosomal subunit.

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

Cited by 16 publications

References 0 publications

Ribosomal protein L7/L12 is required for GTPase translation factors EF‐G, RF3, and IF2 to bind in their GTP state to 70S ribosomes

Ribosomal protein L7/L12 is required for GTPase translation factors EF‐G, RF3, and IF2 to bind in their GTP state to 70S ribosomes

Real-time structural dynamics of late steps in bacterial translation initiation visualized using time-resolved cryogenic electron microscopy

A model for the interaction of the G3‐subdomain of Geobacillus stearothermophilus IF2 with the 30S ribosomal subunit

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