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Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation

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ABSTRACT

In bacterial translational initiation, three initiation factors (IFs 1–3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1–3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNAfMet) into the P site for start codon recognition.

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IF2 in 30S PICs, Related to Figure 6(A) Space filling model of PIC-I, II and III. IF2 is in the same position and conformation in all PICs.(B) Superposition of the crystal structure of Thermus IF2 (PDB: 4KJZ; Eiler et al., 2013) on IF2 in PIC-III shows the large movement of domain C1 (curved arrow) upon binding to the 30S. The small arrow shows the loop after helix8 of C1 domain that interacts with IF1 in this new conformation on 30S.
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figs7: IF2 in 30S PICs, Related to Figure 6(A) Space filling model of PIC-I, II and III. IF2 is in the same position and conformation in all PICs.(B) Superposition of the crystal structure of Thermus IF2 (PDB: 4KJZ; Eiler et al., 2013) on IF2 in PIC-III shows the large movement of domain C1 (curved arrow) upon binding to the 30S. The small arrow shows the loop after helix8 of C1 domain that interacts with IF1 in this new conformation on 30S.

Mentions: The above structures were derived from sample 1, which omitted IF2 in order to be able to characterize the effects of IF2 binding. We also reconstructed EM maps of 30S PIC from another sample 2 containing all IFs including IF2 (Figure S1). The IF2 conformation is found to be similar in all maps (PICs I–III) irrespective of the presence/absence or position of fMet-tRNAfMet in the P site (Figures 6A and S7A). The C2 domain of IF2 is positioned to bind the CCA of fMet-tRNAfMet even prior to its binding, as seen in PIC-I. In PIC-III, the tRNAfMet is accommodated in the P site, and its CCA end with fMet is observed in the pocket of the C2 (Figure 6A). This conformation of IF2 is different from that in the crystal structure of Thermus IF2 (Eiler et al., 2013). In PICs I–III, the rotation of the C1 domain compared to the crystal structure and the conformational change in loop after helix8 of C1 domain allows interaction between IF2 and IF1 (Figure S7B).


Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation
IF2 in 30S PICs, Related to Figure 6(A) Space filling model of PIC-I, II and III. IF2 is in the same position and conformation in all PICs.(B) Superposition of the crystal structure of Thermus IF2 (PDB: 4KJZ; Eiler et al., 2013) on IF2 in PIC-III shows the large movement of domain C1 (curved arrow) upon binding to the 30S. The small arrow shows the loop after helix8 of C1 domain that interacts with IF1 in this new conformation on 30S.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037330&req=5

figs7: IF2 in 30S PICs, Related to Figure 6(A) Space filling model of PIC-I, II and III. IF2 is in the same position and conformation in all PICs.(B) Superposition of the crystal structure of Thermus IF2 (PDB: 4KJZ; Eiler et al., 2013) on IF2 in PIC-III shows the large movement of domain C1 (curved arrow) upon binding to the 30S. The small arrow shows the loop after helix8 of C1 domain that interacts with IF1 in this new conformation on 30S.
Mentions: The above structures were derived from sample 1, which omitted IF2 in order to be able to characterize the effects of IF2 binding. We also reconstructed EM maps of 30S PIC from another sample 2 containing all IFs including IF2 (Figure S1). The IF2 conformation is found to be similar in all maps (PICs I–III) irrespective of the presence/absence or position of fMet-tRNAfMet in the P site (Figures 6A and S7A). The C2 domain of IF2 is positioned to bind the CCA of fMet-tRNAfMet even prior to its binding, as seen in PIC-I. In PIC-III, the tRNAfMet is accommodated in the P site, and its CCA end with fMet is observed in the pocket of the C2 (Figure 6A). This conformation of IF2 is different from that in the crystal structure of Thermus IF2 (Eiler et al., 2013). In PICs I–III, the rotation of the C1 domain compared to the crystal structure and the conformational change in loop after helix8 of C1 domain allows interaction between IF2 and IF1 (Figure S7B).

View Article: PubMed Central - PubMed

ABSTRACT

In bacterial translational initiation, three initiation factors (IFs 1–3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1–3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNAfMet) into the P site for start codon recognition.

No MeSH data available.


Related in: MedlinePlus