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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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Fitting of Ligands in Density Maps, Related to Figure 1(A) Left: Representative snapshot showing side chains of ribosomal protein and rRNA fitting in PIC-1A. Right: Fitting of mRNA (magenta), CTD (orange) and rRNA (yellow) at the P site in PIC-1A.(B) Fitting of mRNA (magenta), CTD (orange) and tRNA (green) at the P site in PIC-2B, PIC-3 and PIC-4.(C) Fitting of mRNA (magenta), IF1 (purple), IF3 (orange) and tRNA (green) in PIC-2A, PIC-3 and PIC-4. IF1 is missing in PIC-4.(D) Fitting of IF2 (blue) and tRNA (green) in PIC-III shown in two orientations. IF1 (purple), CTD (brick red), linker/NTD (orange) are also shown.
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figs3: Fitting of Ligands in Density Maps, Related to Figure 1(A) Left: Representative snapshot showing side chains of ribosomal protein and rRNA fitting in PIC-1A. Right: Fitting of mRNA (magenta), CTD (orange) and rRNA (yellow) at the P site in PIC-1A.(B) Fitting of mRNA (magenta), CTD (orange) and tRNA (green) at the P site in PIC-2B, PIC-3 and PIC-4.(C) Fitting of mRNA (magenta), IF1 (purple), IF3 (orange) and tRNA (green) in PIC-2A, PIC-3 and PIC-4. IF1 is missing in PIC-4.(D) Fitting of IF2 (blue) and tRNA (green) in PIC-III shown in two orientations. IF1 (purple), CTD (brick red), linker/NTD (orange) are also shown.

Mentions: As observed with other ribosomal complexes, the local resolution and the quality of the density are best in the core of the 30S and in ligands directly attached to it (Table S2). In all structures, distinct density is observed for IFs, tRNA, and mRNA; in the higher resolution structures, density for side chains of proteins are clearly visible (Figure S3; Movie S1). IF1 is observed at the A site in a conformation consistent with the previous crystal structure of 30S•IF1 (Carter et al., 2001). In these structures, we see clear dumbbell-shaped density for IF3, consisting of its N-terminal domain (NTD), C-terminal domain (CTD), and the largely helical linker that separates them (Biou et al., 1995). The CTD binds reversibly near the P site, with conserved residues approaching the codon:anticodon helix, while the NTD binds first to the platform and then to the elbow of fMet-tRNAfMet. In each structure, the NTD and CTD occupy different positions near the 30S platform and P site, respectively. Thus, IF3 is observed in distinct conformations in the various 30S PICs, which we propose represent different stages of initiation.


Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation
Fitting of Ligands in Density Maps, Related to Figure 1(A) Left: Representative snapshot showing side chains of ribosomal protein and rRNA fitting in PIC-1A. Right: Fitting of mRNA (magenta), CTD (orange) and rRNA (yellow) at the P site in PIC-1A.(B) Fitting of mRNA (magenta), CTD (orange) and tRNA (green) at the P site in PIC-2B, PIC-3 and PIC-4.(C) Fitting of mRNA (magenta), IF1 (purple), IF3 (orange) and tRNA (green) in PIC-2A, PIC-3 and PIC-4. IF1 is missing in PIC-4.(D) Fitting of IF2 (blue) and tRNA (green) in PIC-III shown in two orientations. IF1 (purple), CTD (brick red), linker/NTD (orange) are also shown.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

figs3: Fitting of Ligands in Density Maps, Related to Figure 1(A) Left: Representative snapshot showing side chains of ribosomal protein and rRNA fitting in PIC-1A. Right: Fitting of mRNA (magenta), CTD (orange) and rRNA (yellow) at the P site in PIC-1A.(B) Fitting of mRNA (magenta), CTD (orange) and tRNA (green) at the P site in PIC-2B, PIC-3 and PIC-4.(C) Fitting of mRNA (magenta), IF1 (purple), IF3 (orange) and tRNA (green) in PIC-2A, PIC-3 and PIC-4. IF1 is missing in PIC-4.(D) Fitting of IF2 (blue) and tRNA (green) in PIC-III shown in two orientations. IF1 (purple), CTD (brick red), linker/NTD (orange) are also shown.
Mentions: As observed with other ribosomal complexes, the local resolution and the quality of the density are best in the core of the 30S and in ligands directly attached to it (Table S2). In all structures, distinct density is observed for IFs, tRNA, and mRNA; in the higher resolution structures, density for side chains of proteins are clearly visible (Figure S3; Movie S1). IF1 is observed at the A site in a conformation consistent with the previous crystal structure of 30S•IF1 (Carter et al., 2001). In these structures, we see clear dumbbell-shaped density for IF3, consisting of its N-terminal domain (NTD), C-terminal domain (CTD), and the largely helical linker that separates them (Biou et al., 1995). The CTD binds reversibly near the P site, with conserved residues approaching the codon:anticodon helix, while the NTD binds first to the platform and then to the elbow of fMet-tRNAfMet. In each structure, the NTD and CTD occupy different positions near the 30S platform and P site, respectively. Thus, IF3 is observed in distinct conformations in the various 30S PICs, which we propose represent different stages of 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.


Related in: MedlinePlus