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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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Validation of the PICs, Related to Figure 1(A) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −1A, 1B and 1C.(B) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −2A, 2B and 2C. (C) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −3 and 4.(D) Gold-standard Fourier Shell Correlation (FSC) curves for PICs -I, II and III.(E) Analysis of overfitting by cross-validation of the PIC-1A model. FSCwork curves (red) corresponding to the refined model versus the half-map it was refined against, and FSCtest curves (green), i.e., those calculated between the refined atomic model and the other half-map. The black curve shows the FSC curve between a reconstruction from all particles and the model refined against the map.(F) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-3 model.(G) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-4 model.(H) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-III model.
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figs2: Validation of the PICs, Related to Figure 1(A) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −1A, 1B and 1C.(B) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −2A, 2B and 2C. (C) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −3 and 4.(D) Gold-standard Fourier Shell Correlation (FSC) curves for PICs -I, II and III.(E) Analysis of overfitting by cross-validation of the PIC-1A model. FSCwork curves (red) corresponding to the refined model versus the half-map it was refined against, and FSCtest curves (green), i.e., those calculated between the refined atomic model and the other half-map. The black curve shows the FSC curve between a reconstruction from all particles and the model refined against the map.(F) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-3 model.(G) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-4 model.(H) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-III model.

Mentions: We have determined single-particle cryoEM structures of the 30S PIC from two samples, respectively with and without IF2 (Figures 1, S1, and S2; Table S1). Maps derived from sample 1 (prepared without IF2) are named from PIC 1–4, with the numbering intended to reflect a plausible order of events in the initiation process (see Methods Details for how this was inferred). PICs 1A–1C contain IF1, IF3, and mRNA (but lack tRNA) and differ only in the conformation of the 30S head. PICs 2A–2C contain IF1, IF3, mRNA, and fMet-tRNAfMet and represent states after binding of tRNA but prior to its full accommodation in the P site. PIC-2A shows the initial binding of tRNA at P site in an open conformation of 30S. PIC-2C shows tRNA in a closed conformation of 30S, while PIC-2B represents a likely intermediate step between PIC-2A and 2C. PIC-3 shows a more engaged fMet-tRNAfMet, while PIC-4 shows a fully accommodated fMet-tRNAfMet at the P site.


Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation
Validation of the PICs, Related to Figure 1(A) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −1A, 1B and 1C.(B) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −2A, 2B and 2C. (C) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −3 and 4.(D) Gold-standard Fourier Shell Correlation (FSC) curves for PICs -I, II and III.(E) Analysis of overfitting by cross-validation of the PIC-1A model. FSCwork curves (red) corresponding to the refined model versus the half-map it was refined against, and FSCtest curves (green), i.e., those calculated between the refined atomic model and the other half-map. The black curve shows the FSC curve between a reconstruction from all particles and the model refined against the map.(F) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-3 model.(G) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-4 model.(H) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-III model.
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figs2: Validation of the PICs, Related to Figure 1(A) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −1A, 1B and 1C.(B) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −2A, 2B and 2C. (C) Gold-standard Fourier Shell Correlation (FSC) curves for PICs −3 and 4.(D) Gold-standard Fourier Shell Correlation (FSC) curves for PICs -I, II and III.(E) Analysis of overfitting by cross-validation of the PIC-1A model. FSCwork curves (red) corresponding to the refined model versus the half-map it was refined against, and FSCtest curves (green), i.e., those calculated between the refined atomic model and the other half-map. The black curve shows the FSC curve between a reconstruction from all particles and the model refined against the map.(F) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-3 model.(G) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-4 model.(H) Analysis of overfitting by cross-validation, similar to that in (E), of the PIC-III model.
Mentions: We have determined single-particle cryoEM structures of the 30S PIC from two samples, respectively with and without IF2 (Figures 1, S1, and S2; Table S1). Maps derived from sample 1 (prepared without IF2) are named from PIC 1–4, with the numbering intended to reflect a plausible order of events in the initiation process (see Methods Details for how this was inferred). PICs 1A–1C contain IF1, IF3, and mRNA (but lack tRNA) and differ only in the conformation of the 30S head. PICs 2A–2C contain IF1, IF3, mRNA, and fMet-tRNAfMet and represent states after binding of tRNA but prior to its full accommodation in the P site. PIC-2A shows the initial binding of tRNA at P site in an open conformation of 30S. PIC-2C shows tRNA in a closed conformation of 30S, while PIC-2B represents a likely intermediate step between PIC-2A and 2C. PIC-3 shows a more engaged fMet-tRNAfMet, while PIC-4 shows a fully accommodated fMet-tRNAfMet at the P site.

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