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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.

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Related in: MedlinePlus

tRNA and IF3 in PICs, Related to Figure 5(A) The relative movement of the initiator tRNA in the various PICs as deduced by a superposition using the 30S body. The tip of the ASL in PIC-2A (purple) is about 3 Å away from that of PIC-2C (cyan), PIC-3 (green) and PIC-4 (red) while the tip of the ASL in PIC-2B (yellow) is about 7 Å away from the rest. Top Inset: Superposition of PIC-2C (cyan), PIC-3 (green) and PIC-4 (red) using 16S rRNA of the 30S body shows the tilting of the tRNA toward the body. The clashes of the CTD in Position1 (cyan) and Position1′ (green outline) with the tRNAs are also highlighted by arrows. Bottom Inset: tRNA in PIC-4 (red) and in PIC-2B (yellow) are shown with respect to canonical P/P (blue) and E/E (pale blue) tRNAs in the 70S complex, superimposed based on the 30S body. The tRNA in PIC-2B is positioned between the P and E site tRNAs.(B) Interactions of IF3 with the mRNA at the P site in PIC-2C. Arg159 (part of β-hairpin) is in close proximity to ASL. Arg123 of CTD interacts with +4 base of the mRNA. Arg91 and Lys93 of CTD are in vicinity and are also shown.(C) Interactions of IF3 with the mRNA at the P site in PIC-3. Arg159 (part of β-hairpin) is in close proximity to ASL. Arg123 of CTD is also shown. uS9 and uS13 tails interact with ASL.(D) Surface representation of tRNA and IF3 in PIC-3. The CTD and linker is positioned very close to ASL making interactions with it while NTD interacts with elbow of tRNA.(E) tRNA in PIC-2B moves ∼7 Å toward the E site (dashed arrow). The P-site tRNA is shown in transparent gray to highlight the movement. The movement places the tRNA in PIC-2B away from the CTD at the P site.(F) Superposition using the 30S head of PIC-2B (gray) and PIC-4 (color) shows that the codon:anticodon helices superimpose very well at the P site. This shows that the movement of codon:anticodon helix toward the E site in PIC-2B is due to its unique 30S head swivel.
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figs6: tRNA and IF3 in PICs, Related to Figure 5(A) The relative movement of the initiator tRNA in the various PICs as deduced by a superposition using the 30S body. The tip of the ASL in PIC-2A (purple) is about 3 Å away from that of PIC-2C (cyan), PIC-3 (green) and PIC-4 (red) while the tip of the ASL in PIC-2B (yellow) is about 7 Å away from the rest. Top Inset: Superposition of PIC-2C (cyan), PIC-3 (green) and PIC-4 (red) using 16S rRNA of the 30S body shows the tilting of the tRNA toward the body. The clashes of the CTD in Position1 (cyan) and Position1′ (green outline) with the tRNAs are also highlighted by arrows. Bottom Inset: tRNA in PIC-4 (red) and in PIC-2B (yellow) are shown with respect to canonical P/P (blue) and E/E (pale blue) tRNAs in the 70S complex, superimposed based on the 30S body. The tRNA in PIC-2B is positioned between the P and E site tRNAs.(B) Interactions of IF3 with the mRNA at the P site in PIC-2C. Arg159 (part of β-hairpin) is in close proximity to ASL. Arg123 of CTD interacts with +4 base of the mRNA. Arg91 and Lys93 of CTD are in vicinity and are also shown.(C) Interactions of IF3 with the mRNA at the P site in PIC-3. Arg159 (part of β-hairpin) is in close proximity to ASL. Arg123 of CTD is also shown. uS9 and uS13 tails interact with ASL.(D) Surface representation of tRNA and IF3 in PIC-3. The CTD and linker is positioned very close to ASL making interactions with it while NTD interacts with elbow of tRNA.(E) tRNA in PIC-2B moves ∼7 Å toward the E site (dashed arrow). The P-site tRNA is shown in transparent gray to highlight the movement. The movement places the tRNA in PIC-2B away from the CTD at the P site.(F) Superposition using the 30S head of PIC-2B (gray) and PIC-4 (color) shows that the codon:anticodon helices superimpose very well at the P site. This shows that the movement of codon:anticodon helix toward the E site in PIC-2B is due to its unique 30S head swivel.

Mentions: The tRNA in closed conformation of PIC-2C moves closer to the body with the narrowing of the P site compared to PIC-2A. The tip of the ASL in PIC-2C is positioned about 3 Å from that seen in the PIC-2A complex (Figure S6A). Also in PIC-2C, its ASL is tilted toward the body (Figure S6A), and it comes very close to CTD and only a small movement of β-hairpin away from the fMet-tRNAfMet is required to keep CTD in position 1 (Figure 5C). Arg123 of CTD H-bonds with +4 base of mRNA, which may help to keep the mRNA in position, or it might play an indirect role in codon:anticodon discrimination (Figure S6B).


Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation
tRNA and IF3 in PICs, Related to Figure 5(A) The relative movement of the initiator tRNA in the various PICs as deduced by a superposition using the 30S body. The tip of the ASL in PIC-2A (purple) is about 3 Å away from that of PIC-2C (cyan), PIC-3 (green) and PIC-4 (red) while the tip of the ASL in PIC-2B (yellow) is about 7 Å away from the rest. Top Inset: Superposition of PIC-2C (cyan), PIC-3 (green) and PIC-4 (red) using 16S rRNA of the 30S body shows the tilting of the tRNA toward the body. The clashes of the CTD in Position1 (cyan) and Position1′ (green outline) with the tRNAs are also highlighted by arrows. Bottom Inset: tRNA in PIC-4 (red) and in PIC-2B (yellow) are shown with respect to canonical P/P (blue) and E/E (pale blue) tRNAs in the 70S complex, superimposed based on the 30S body. The tRNA in PIC-2B is positioned between the P and E site tRNAs.(B) Interactions of IF3 with the mRNA at the P site in PIC-2C. Arg159 (part of β-hairpin) is in close proximity to ASL. Arg123 of CTD interacts with +4 base of the mRNA. Arg91 and Lys93 of CTD are in vicinity and are also shown.(C) Interactions of IF3 with the mRNA at the P site in PIC-3. Arg159 (part of β-hairpin) is in close proximity to ASL. Arg123 of CTD is also shown. uS9 and uS13 tails interact with ASL.(D) Surface representation of tRNA and IF3 in PIC-3. The CTD and linker is positioned very close to ASL making interactions with it while NTD interacts with elbow of tRNA.(E) tRNA in PIC-2B moves ∼7 Å toward the E site (dashed arrow). The P-site tRNA is shown in transparent gray to highlight the movement. The movement places the tRNA in PIC-2B away from the CTD at the P site.(F) Superposition using the 30S head of PIC-2B (gray) and PIC-4 (color) shows that the codon:anticodon helices superimpose very well at the P site. This shows that the movement of codon:anticodon helix toward the E site in PIC-2B is due to its unique 30S head swivel.
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figs6: tRNA and IF3 in PICs, Related to Figure 5(A) The relative movement of the initiator tRNA in the various PICs as deduced by a superposition using the 30S body. The tip of the ASL in PIC-2A (purple) is about 3 Å away from that of PIC-2C (cyan), PIC-3 (green) and PIC-4 (red) while the tip of the ASL in PIC-2B (yellow) is about 7 Å away from the rest. Top Inset: Superposition of PIC-2C (cyan), PIC-3 (green) and PIC-4 (red) using 16S rRNA of the 30S body shows the tilting of the tRNA toward the body. The clashes of the CTD in Position1 (cyan) and Position1′ (green outline) with the tRNAs are also highlighted by arrows. Bottom Inset: tRNA in PIC-4 (red) and in PIC-2B (yellow) are shown with respect to canonical P/P (blue) and E/E (pale blue) tRNAs in the 70S complex, superimposed based on the 30S body. The tRNA in PIC-2B is positioned between the P and E site tRNAs.(B) Interactions of IF3 with the mRNA at the P site in PIC-2C. Arg159 (part of β-hairpin) is in close proximity to ASL. Arg123 of CTD interacts with +4 base of the mRNA. Arg91 and Lys93 of CTD are in vicinity and are also shown.(C) Interactions of IF3 with the mRNA at the P site in PIC-3. Arg159 (part of β-hairpin) is in close proximity to ASL. Arg123 of CTD is also shown. uS9 and uS13 tails interact with ASL.(D) Surface representation of tRNA and IF3 in PIC-3. The CTD and linker is positioned very close to ASL making interactions with it while NTD interacts with elbow of tRNA.(E) tRNA in PIC-2B moves ∼7 Å toward the E site (dashed arrow). The P-site tRNA is shown in transparent gray to highlight the movement. The movement places the tRNA in PIC-2B away from the CTD at the P site.(F) Superposition using the 30S head of PIC-2B (gray) and PIC-4 (color) shows that the codon:anticodon helices superimpose very well at the P site. This shows that the movement of codon:anticodon helix toward the E site in PIC-2B is due to its unique 30S head swivel.
Mentions: The tRNA in closed conformation of PIC-2C moves closer to the body with the narrowing of the P site compared to PIC-2A. The tip of the ASL in PIC-2C is positioned about 3 Å from that seen in the PIC-2A complex (Figure S6A). Also in PIC-2C, its ASL is tilted toward the body (Figure S6A), and it comes very close to CTD and only a small movement of β-hairpin away from the fMet-tRNAfMet is required to keep CTD in position 1 (Figure 5C). Arg123 of CTD H-bonds with +4 base of mRNA, which may help to keep the mRNA in position, or it might play an indirect role in codon:anticodon discrimination (Figure S6B).

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