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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.


Three Distinct Conformations of IF3 on 30S PICs(A) In PIC-2A, the CTD is in position 1 at the P site, while the NTD moves away (arrow; NTD movement is measured using Arg36 as the reference point) from the platform to bind to the elbow of the fMet-tRNAfMet. The linker remains attached to h23 and h24. IF3 from PIC-1C is shown in gray for comparison. Inset shows the movement of the β-hairpin (using Arg159 as the reference) with respect to that in PIC-1C.(B) In PIC-3, the CTD moves slightly to position 1′ at the P site and the NTD moves ∼27 Å while attached to the elbow of the fMet-tRNAfMet. The linker is no longer in contact with 30S. IF3 from PIC-2A is shown in gray for comparison. Inset shows the movement of β-hairpin with respect to that in PIC-2A.(C) In PIC-4, the CTD moves ∼25 Å away from the P site to position 2, while the NTD moves while attached to the elbow of the fMet-tRNAfMet. The linker is no longer in contact with 30S. IF3 from PIC-3 is shown in gray for comparison. IF1 is not present in PIC-4, but its location in PIC-3 is shown here to highlight the movement of the CTD away from it.(D) Superposition of IF3 in PIC-1C (yellow), PIC-2A (cyan), PIC-3 (orange), PIC-4 (red). The fMet-tRNAfMet and IF1 from PIC-3 are shown to highlight the different conformations of CTD and NTD. The movement of the CTD is measured using Arg125 as the reference point. The arrows show the overall direction of movement of the two domains.See also Figure S4 and Movie S3.
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fig4: Three Distinct Conformations of IF3 on 30S PICs(A) In PIC-2A, the CTD is in position 1 at the P site, while the NTD moves away (arrow; NTD movement is measured using Arg36 as the reference point) from the platform to bind to the elbow of the fMet-tRNAfMet. The linker remains attached to h23 and h24. IF3 from PIC-1C is shown in gray for comparison. Inset shows the movement of the β-hairpin (using Arg159 as the reference) with respect to that in PIC-1C.(B) In PIC-3, the CTD moves slightly to position 1′ at the P site and the NTD moves ∼27 Å while attached to the elbow of the fMet-tRNAfMet. The linker is no longer in contact with 30S. IF3 from PIC-2A is shown in gray for comparison. Inset shows the movement of β-hairpin with respect to that in PIC-2A.(C) In PIC-4, the CTD moves ∼25 Å away from the P site to position 2, while the NTD moves while attached to the elbow of the fMet-tRNAfMet. The linker is no longer in contact with 30S. IF3 from PIC-3 is shown in gray for comparison. IF1 is not present in PIC-4, but its location in PIC-3 is shown here to highlight the movement of the CTD away from it.(D) Superposition of IF3 in PIC-1C (yellow), PIC-2A (cyan), PIC-3 (orange), PIC-4 (red). The fMet-tRNAfMet and IF1 from PIC-3 are shown to highlight the different conformations of CTD and NTD. The movement of the CTD is measured using Arg125 as the reference point. The arrows show the overall direction of movement of the two domains.See also Figure S4 and Movie S3.

Mentions: Once tRNA binds, both domains of IF3 appear to sample different positions. Upon binding of fMet-tRNAfMet to an open 30S conformation (PIC-2A), the IF3 NTD moves 9 Å away from the platform to interact with the elbow of tRNA, while the CTD remains in its position at the P site (Figure 4A; Movie S3). Since the open conformation of 30S has a widened P site, the tRNA is not completely engaged in the P site and hence does not have a steric clash with the CTD; only a subtle movement of ∼1.5 Å of the CTD β-hairpin away from the tRNA is seen. The linker too remains almost unchanged.


Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation
Three Distinct Conformations of IF3 on 30S PICs(A) In PIC-2A, the CTD is in position 1 at the P site, while the NTD moves away (arrow; NTD movement is measured using Arg36 as the reference point) from the platform to bind to the elbow of the fMet-tRNAfMet. The linker remains attached to h23 and h24. IF3 from PIC-1C is shown in gray for comparison. Inset shows the movement of the β-hairpin (using Arg159 as the reference) with respect to that in PIC-1C.(B) In PIC-3, the CTD moves slightly to position 1′ at the P site and the NTD moves ∼27 Å while attached to the elbow of the fMet-tRNAfMet. The linker is no longer in contact with 30S. IF3 from PIC-2A is shown in gray for comparison. Inset shows the movement of β-hairpin with respect to that in PIC-2A.(C) In PIC-4, the CTD moves ∼25 Å away from the P site to position 2, while the NTD moves while attached to the elbow of the fMet-tRNAfMet. The linker is no longer in contact with 30S. IF3 from PIC-3 is shown in gray for comparison. IF1 is not present in PIC-4, but its location in PIC-3 is shown here to highlight the movement of the CTD away from it.(D) Superposition of IF3 in PIC-1C (yellow), PIC-2A (cyan), PIC-3 (orange), PIC-4 (red). The fMet-tRNAfMet and IF1 from PIC-3 are shown to highlight the different conformations of CTD and NTD. The movement of the CTD is measured using Arg125 as the reference point. The arrows show the overall direction of movement of the two domains.See also Figure S4 and Movie S3.
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fig4: Three Distinct Conformations of IF3 on 30S PICs(A) In PIC-2A, the CTD is in position 1 at the P site, while the NTD moves away (arrow; NTD movement is measured using Arg36 as the reference point) from the platform to bind to the elbow of the fMet-tRNAfMet. The linker remains attached to h23 and h24. IF3 from PIC-1C is shown in gray for comparison. Inset shows the movement of the β-hairpin (using Arg159 as the reference) with respect to that in PIC-1C.(B) In PIC-3, the CTD moves slightly to position 1′ at the P site and the NTD moves ∼27 Å while attached to the elbow of the fMet-tRNAfMet. The linker is no longer in contact with 30S. IF3 from PIC-2A is shown in gray for comparison. Inset shows the movement of β-hairpin with respect to that in PIC-2A.(C) In PIC-4, the CTD moves ∼25 Å away from the P site to position 2, while the NTD moves while attached to the elbow of the fMet-tRNAfMet. The linker is no longer in contact with 30S. IF3 from PIC-3 is shown in gray for comparison. IF1 is not present in PIC-4, but its location in PIC-3 is shown here to highlight the movement of the CTD away from it.(D) Superposition of IF3 in PIC-1C (yellow), PIC-2A (cyan), PIC-3 (orange), PIC-4 (red). The fMet-tRNAfMet and IF1 from PIC-3 are shown to highlight the different conformations of CTD and NTD. The movement of the CTD is measured using Arg125 as the reference point. The arrows show the overall direction of movement of the two domains.See also Figure S4 and Movie S3.
Mentions: Once tRNA binds, both domains of IF3 appear to sample different positions. Upon binding of fMet-tRNAfMet to an open 30S conformation (PIC-2A), the IF3 NTD moves 9 Å away from the platform to interact with the elbow of tRNA, while the CTD remains in its position at the P site (Figure 4A; Movie S3). Since the open conformation of 30S has a widened P site, the tRNA is not completely engaged in the P site and hence does not have a steric clash with the CTD; only a subtle movement of ∼1.5 Å of the CTD β-hairpin away from the tRNA is seen. The linker too remains almost unchanged.

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.