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

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.


IF2 in 30S PICs(A) Ribbon representation of IF2 in PIC-III highlighting the four domains of IF2 seen in the structures. Each domain is shown is in different shades of blue and labeled. The C-terminal domain C2 interacts with the acceptor arm of the fMet-tRNAfMet. The fMet moiety is shown as spheres in its known pocket in C2.(B) Superposition of IF2 fit into previous lower-resolution 30S initiation complex structures (30S IC, gray; Simonetti et al., 2013) with IF2 in PIC-III, showing that the overall position and conformation of IF2 are similar. However, marked differences are seen in the orientation of the C2 domain, the loop connecting helix 8 and domain C1 (that interacts with IF1) and a loop in the G domain. The arrows indicate these differences.(C) Comparison of IF2 in PIC-III (shades of blue) with that in the 70S complex (pink; Sprink et al., 2016) shows a similar position and conformation for most domains of IF2 except C2. The arrow shows that in the 70S there is a movement of C2 toward the 30S body to avoid a clash with 50S (shown in gray). IF1, CTD of IF3 and acceptor arm of fMet-tRNAfMet from PIC-III is also shown.See also Figure S7 and Movie S5.
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fig6: IF2 in 30S PICs(A) Ribbon representation of IF2 in PIC-III highlighting the four domains of IF2 seen in the structures. Each domain is shown is in different shades of blue and labeled. The C-terminal domain C2 interacts with the acceptor arm of the fMet-tRNAfMet. The fMet moiety is shown as spheres in its known pocket in C2.(B) Superposition of IF2 fit into previous lower-resolution 30S initiation complex structures (30S IC, gray; Simonetti et al., 2013) with IF2 in PIC-III, showing that the overall position and conformation of IF2 are similar. However, marked differences are seen in the orientation of the C2 domain, the loop connecting helix 8 and domain C1 (that interacts with IF1) and a loop in the G domain. The arrows indicate these differences.(C) Comparison of IF2 in PIC-III (shades of blue) with that in the 70S complex (pink; Sprink et al., 2016) shows a similar position and conformation for most domains of IF2 except C2. The arrow shows that in the 70S there is a movement of C2 toward the 30S body to avoid a clash with 50S (shown in gray). IF1, CTD of IF3 and acceptor arm of fMet-tRNAfMet from PIC-III is also shown.See also Figure S7 and Movie S5.

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(A) Ribbon representation of IF2 in PIC-III highlighting the four domains of IF2 seen in the structures. Each domain is shown is in different shades of blue and labeled. The C-terminal domain C2 interacts with the acceptor arm of the fMet-tRNAfMet. The fMet moiety is shown as spheres in its known pocket in C2.(B) Superposition of IF2 fit into previous lower-resolution 30S initiation complex structures (30S IC, gray; Simonetti et al., 2013) with IF2 in PIC-III, showing that the overall position and conformation of IF2 are similar. However, marked differences are seen in the orientation of the C2 domain, the loop connecting helix 8 and domain C1 (that interacts with IF1) and a loop in the G domain. The arrows indicate these differences.(C) Comparison of IF2 in PIC-III (shades of blue) with that in the 70S complex (pink; Sprink et al., 2016) shows a similar position and conformation for most domains of IF2 except C2. The arrow shows that in the 70S there is a movement of C2 toward the 30S body to avoid a clash with 50S (shown in gray). IF1, CTD of IF3 and acceptor arm of fMet-tRNAfMet from PIC-III is also shown.See also Figure S7 and Movie S5.
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fig6: IF2 in 30S PICs(A) Ribbon representation of IF2 in PIC-III highlighting the four domains of IF2 seen in the structures. Each domain is shown is in different shades of blue and labeled. The C-terminal domain C2 interacts with the acceptor arm of the fMet-tRNAfMet. The fMet moiety is shown as spheres in its known pocket in C2.(B) Superposition of IF2 fit into previous lower-resolution 30S initiation complex structures (30S IC, gray; Simonetti et al., 2013) with IF2 in PIC-III, showing that the overall position and conformation of IF2 are similar. However, marked differences are seen in the orientation of the C2 domain, the loop connecting helix 8 and domain C1 (that interacts with IF1) and a loop in the G domain. The arrows indicate these differences.(C) Comparison of IF2 in PIC-III (shades of blue) with that in the 70S complex (pink; Sprink et al., 2016) shows a similar position and conformation for most domains of IF2 except C2. The arrow shows that in the 70S there is a movement of C2 toward the 30S body to avoid a clash with 50S (shown in gray). IF1, CTD of IF3 and acceptor arm of fMet-tRNAfMet from PIC-III is also shown.See also Figure S7 and Movie S5.
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.