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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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Scheme of 3D Classification of Data, Related to Figure 1(A) For Sample1 (without IF2) 666,610 particles were selected after 2D classification and an initial 3D refinement was done. The ‘ligands mask’ used for focused 3D classification is shown by an outline. The scheme shows how each PIC was obtained. (See ‘Image processing and structure determination’ in Methods Details for detailed information).(B) For Sample2 (with IF2) 803,433 particles were selected after 2D classification and an initial 3D refinement was done. The ‘IF2 mask’ as well as ‘ligands mask’ used for focused 3D classification is shown by an outline. The scheme shows how PICs- I to III were obtained. (See ‘Image processing and structure determination’ in Methods Details for detailed information).
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figs1: Scheme of 3D Classification of Data, Related to Figure 1(A) For Sample1 (without IF2) 666,610 particles were selected after 2D classification and an initial 3D refinement was done. The ‘ligands mask’ used for focused 3D classification is shown by an outline. The scheme shows how each PIC was obtained. (See ‘Image processing and structure determination’ in Methods Details for detailed information).(B) For Sample2 (with IF2) 803,433 particles were selected after 2D classification and an initial 3D refinement was done. The ‘IF2 mask’ as well as ‘ligands mask’ used for focused 3D classification is shown by an outline. The scheme shows how PICs- I to III were obtained. (See ‘Image processing and structure determination’ in Methods Details for detailed information).

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
Scheme of 3D Classification of Data, Related to Figure 1(A) For Sample1 (without IF2) 666,610 particles were selected after 2D classification and an initial 3D refinement was done. The ‘ligands mask’ used for focused 3D classification is shown by an outline. The scheme shows how each PIC was obtained. (See ‘Image processing and structure determination’ in Methods Details for detailed information).(B) For Sample2 (with IF2) 803,433 particles were selected after 2D classification and an initial 3D refinement was done. The ‘IF2 mask’ as well as ‘ligands mask’ used for focused 3D classification is shown by an outline. The scheme shows how PICs- I to III were obtained. (See ‘Image processing and structure determination’ in Methods Details for detailed information).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037330&req=5

figs1: Scheme of 3D Classification of Data, Related to Figure 1(A) For Sample1 (without IF2) 666,610 particles were selected after 2D classification and an initial 3D refinement was done. The ‘ligands mask’ used for focused 3D classification is shown by an outline. The scheme shows how each PIC was obtained. (See ‘Image processing and structure determination’ in Methods Details for detailed information).(B) For Sample2 (with IF2) 803,433 particles were selected after 2D classification and an initial 3D refinement was done. The ‘IF2 mask’ as well as ‘ligands mask’ used for focused 3D classification is shown by an outline. The scheme shows how PICs- I to III were obtained. (See ‘Image processing and structure determination’ in Methods Details for detailed information).
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