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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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Movement of the 30S Head Widens the mRNA Entry Channel and Opens the Latch(A) Front view of the orientation of the 30S head in PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red), using the 30S body for alignment. The displacement of the beak in the three PICs highlights the difference in the orientation of the head. The P-site tRNA is shown. The top view shows the rotation axis of the head around h28. The magnitudes of the changes are shown using C1030b as the reference point.(B) Front view of superposition of refined models of PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red) indicating elements forming the latch. The movement of 12 Å of A1001 in the 30S head is shown. Bottom panel: surface representation of PICs 1C, 2B, and 4, with the arrows indicating the opening and closing of the mRNA latch.(C) The conformation of h28 in PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red). Equivalent atoms in h28 (C1384) are shown as spheres. In PIC-1C, h28 is not compressed, and the mRNA latch is open. The opposite is seen in PIC-4. In PIC-2B, the mRNA latch is closed by a head swivel rather than compression of h28.See also Table S3 and Movie S2.
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fig2: Movement of the 30S Head Widens the mRNA Entry Channel and Opens the Latch(A) Front view of the orientation of the 30S head in PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red), using the 30S body for alignment. The displacement of the beak in the three PICs highlights the difference in the orientation of the head. The P-site tRNA is shown. The top view shows the rotation axis of the head around h28. The magnitudes of the changes are shown using C1030b as the reference point.(B) Front view of superposition of refined models of PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red) indicating elements forming the latch. The movement of 12 Å of A1001 in the 30S head is shown. Bottom panel: surface representation of PICs 1C, 2B, and 4, with the arrows indicating the opening and closing of the mRNA latch.(C) The conformation of h28 in PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red). Equivalent atoms in h28 (C1384) are shown as spheres. In PIC-1C, h28 is not compressed, and the mRNA latch is open. The opposite is seen in PIC-4. In PIC-2B, the mRNA latch is closed by a head swivel rather than compression of h28.See also Table S3 and Movie S2.

Mentions: A comparison of all 30S PICs presented here shows various positions of the head of 30S with respect to the body, with motions best described as swiveling and/or nodding of the head. An overall rotation of 8–9° of the 30S head along 16S rRNA helix 28 (h28), which forms the “neck” connecting head to the body, is observed among the complexes (Figure 2A; Movie S2). The 30S head is observed with the same canonical swivel seen in 70S ribosomes (Selmer et al., 2006) or eukaryotic initiation complexes (Hussain et al., 2014, Llácer et al., 2015) in most complexes, namely, PICs 1A, 2C, 3, and 4 and PICs I and III (Table S3). In contrast, the 30S head is swiveled in PICs 1B and 2B. The rotation of the head leads to rearrangement of rRNA at the P site as well as movement of the codon:anticodon helix.


Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation
Movement of the 30S Head Widens the mRNA Entry Channel and Opens the Latch(A) Front view of the orientation of the 30S head in PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red), using the 30S body for alignment. The displacement of the beak in the three PICs highlights the difference in the orientation of the head. The P-site tRNA is shown. The top view shows the rotation axis of the head around h28. The magnitudes of the changes are shown using C1030b as the reference point.(B) Front view of superposition of refined models of PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red) indicating elements forming the latch. The movement of 12 Å of A1001 in the 30S head is shown. Bottom panel: surface representation of PICs 1C, 2B, and 4, with the arrows indicating the opening and closing of the mRNA latch.(C) The conformation of h28 in PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red). Equivalent atoms in h28 (C1384) are shown as spheres. In PIC-1C, h28 is not compressed, and the mRNA latch is open. The opposite is seen in PIC-4. In PIC-2B, the mRNA latch is closed by a head swivel rather than compression of h28.See also Table S3 and Movie S2.
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fig2: Movement of the 30S Head Widens the mRNA Entry Channel and Opens the Latch(A) Front view of the orientation of the 30S head in PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red), using the 30S body for alignment. The displacement of the beak in the three PICs highlights the difference in the orientation of the head. The P-site tRNA is shown. The top view shows the rotation axis of the head around h28. The magnitudes of the changes are shown using C1030b as the reference point.(B) Front view of superposition of refined models of PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red) indicating elements forming the latch. The movement of 12 Å of A1001 in the 30S head is shown. Bottom panel: surface representation of PICs 1C, 2B, and 4, with the arrows indicating the opening and closing of the mRNA latch.(C) The conformation of h28 in PIC-1C (yellow), PIC-2B (blue), and PIC-4 (red). Equivalent atoms in h28 (C1384) are shown as spheres. In PIC-1C, h28 is not compressed, and the mRNA latch is open. The opposite is seen in PIC-4. In PIC-2B, the mRNA latch is closed by a head swivel rather than compression of h28.See also Table S3 and Movie S2.
Mentions: A comparison of all 30S PICs presented here shows various positions of the head of 30S with respect to the body, with motions best described as swiveling and/or nodding of the head. An overall rotation of 8–9° of the 30S head along 16S rRNA helix 28 (h28), which forms the “neck” connecting head to the body, is observed among the complexes (Figure 2A; Movie S2). The 30S head is observed with the same canonical swivel seen in 70S ribosomes (Selmer et al., 2006) or eukaryotic initiation complexes (Hussain et al., 2014, Llácer et al., 2015) in most complexes, namely, PICs 1A, 2C, 3, and 4 and PICs I and III (Table S3). In contrast, the 30S head is swiveled in PICs 1B and 2B. The rotation of the head leads to rearrangement of rRNA at the P site as well as movement of the codon:anticodon helix.

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