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, 36 (4), 475-486

The Complete Structure of the Chloroplast 70S Ribosome in Complex With Translation Factor pY

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The Complete Structure of the Chloroplast 70S Ribosome in Complex With Translation Factor pY

Philipp Bieri et al. EMBO J.

Abstract

Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo-EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid-specific ribosomal proteins. These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid-specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light- and temperature-dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A-site and P-site, where it protects the decoding centre and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non-rotated state, in which the intersubunit bridges to the large subunit are stabilized.

Keywords: chloroplast; cryo‐EM; pY; ribosome; translation.

Figures

Figure 1
Figure 1. Architecture of the chloroplast 70S ribosome

Structure of the chloroplast 70S ribosome. 50S subunit proteins are in blue, 23S rRNA in cyan, 5S rRNA in green, 4.5S rRNA in red, 30S subunit proteins in gold, 16S rRNA in pale yellow, E‐site tRNA in pink and translation factor pY in green. Plastid‐specific ribosomal proteins cS22, cS23, bTHXc, cL37 and cL38 are shown in red.

Protein and rRNA elements conserved between chloroplast and bacterial 70S ribosome are in blue and grey, respectively. Chloroplast‐specific rRNA elements are shown in purple. Plastid‐specific ribosomal proteins and additional protein extensions are in red and yellow, respectively. Translation factor pY is shown in green. Structural landmarks of the 70S ribosome are indicated.

Figure EV1
Figure EV1. Resolution and quality of the cryo‐EM reconstructions

Surface rendering of the final high‐resolution maps of the 30S subunit (A), the 70S ribosome (B) and the 50S subunit (C).

Local resolution plots showing the surface (D–F) and a cross section (G–I) of the cryo‐EM maps. Local resolution maps of the 30S (D, G), 70S (E, H) and 50S (F, I) are shown from the same view as in panels (A, B and C), respectively.

Fourier shell correlation (FSC) curves of the 30S (J), the 70S (K) and 50S (L) cryo‐EM reconstructions. The indicated resolutions are according to the FSC = 0.143 criterion (“gold‐standard”).

Examples for the quality of the density: (M) ribosomal protein (salmon) interacting with rRNA (grey), (N) protein α‐helix (yellow) and (O) protein β‐sheet (green).

Figure 2
Figure 2. Plastid‐specific ribosomal proteins

De novo built and refined structures of the plastid‐specific ribosomal proteins cL37 (A), cL38 (B) and bTHXc (C) are shown in red, with N‐ and C‐termini indicated. 23S and 16S rRNAs in grey and 5S rRNA in green. Alterations in rRNA elements in comparison with bacteria are indicated with dark colour.

Rigid body fitted models of plastid‐specific ribosomal proteins cS22 and cS23 in red. Helices h6, h10 and h17 of 16S rRNA are indicated.

Figure EV2
Figure EV2. De novo built plastid‐specific ribosomal proteins

The density indicates clear side chain features and allows unambiguous tracing of cL37 (A), cL38 (B) and bTHXc (C).

Binding sites of cL37 (D), cL38 (E) and bTHXc (F) in the chloroplast 70S ribosome.

Corresponding sites to panels (D, E and F), respectively, in the bacterial 70S ribosome (PDB 4YBB; Noeske et al, 2015).

Figure EV3
Figure EV3. The chloroplast 4.5S ribosomal RNA

Primary transcript of the chloroplast rRNA operon. The canonical sequences of ribosomal RNAs are indicated by coloured boxes. The 4.5S rRNA is separated from the 23S rRNA by a 115‐nucleotide RNA spacer.

Comparison of the chloroplast 50S subunit (B) with the bacterial 50S subunit (PDB 4YBB; Noeske et al, 2015) (C) indicating the structural rearrangement of the ribosomal proteins that interact with the 23S rRNA (blue) and the 4.5S rRNA (red).

Secondary structure diagram of the chloroplast 4.5S rRNA (D) and the 3′ end of the bacterial 23S rRNA (E). The interactions of the 5′ and 3′ ends of the 4.5S rRNA with the 23S rRNA are shown. Watson–Crick base pairs are indicated by lines (‐), G•U base pairs by dots (•) and non‐standard base pairs by rings (○).

Model of the chloroplast 4.5S rRNA (F) and the 3′ end of the bacterial 23S rRNA (G). The same nucleotides are shown in the models as represented in the secondary structure diagrams (D, E).

Figure 3
Figure 3. The 4.5S ribosomal RNA and its interactions with ribosomal proteins

Position of the 4.5S rRNA (red) at the surface of the 50S large subunit. The 3′ and 5′ ends of the 4.5S rRNA and the 23S rRNA (blue) are labelled. Ribosomal proteins interacting with or in close proximity to the 4.5S rRNA are shown in different colours.

Stabilization of the 5′ end of the 4.5S rRNA and the 3′ end of the 23S rRNA by the plastid‐specific N‐terminal tail of uL13c (yellow). Specific residues of uL13c, uL22c and 4.5S rRNA are labelled.

Figure EV4
Figure EV4. The hidden breaks of the 23S rRNA

Analysis of ribosomal RNA by agarose gel electrophoresis. RNA was extracted from chloroplast 70S ribosome sample and separated on a 2% (w/v) agarose gel (L: high range RNA ladder; 70S: RNA of chloroplast 70S sample).

Schematic of rRNA processing and assembly in the chloroplast. Because of two specific cleavage sites on the 23S rRNA, called “hidden breaks”, the 23S rRNA gets separated into three fragments: A (0.5 kb), B (1.2 kb) and C (1.1 kb).

Views of the 30S (C) and the 50S subunits (D) from the subunit interface and of the 50S subunit (E) from the solvent accessible side. The rRNA is shown as spheres and coloured according to the elements indicated in panel (B). The positions of the hidden breaks on the 23S rRNA are marked with triangles.

The hidden break indicated with a triangle between fragments A and B is introduced in the connection between helices H2 and H24.

The hidden break indicated with a triangle between fragments B and C is positioned at the stem loop of helix H63. The binding site of the helicase RH39 on helix H62 is coloured black. The electron density map shown in panels (H and K) is low‐pass filtered to 4 Å, and the nucleotides at the hidden break sites are labelled. The exact positions of the hidden breaks on the 23S rRNA sequence shown in panels (F and I) were stated in a previous publication (Liu et al, 2015).

Figure 4
Figure 4. Architecture of the mRNA entry and exit site

Surface representation of the solvent accessible side of the 30S small subunit. Ribosomal proteins bS1c (brown), uS2c (blue), uS3c (green), uS4c (cyan) and uS5c (red) are labelled, and plastid‐specific elements are indicated by darker colour shades.

Helical extensions of bS1c cluster around uS2c.

Chloroplast‐specific extensions of uS4c and uS5c remodel the mRNA entry site (marked with an asterisk).

Figure 5
Figure 5. Architecture of the polypeptide tunnel exit

Surface representation of the polypeptide exit site (PES) of the 50S large subunit. Ribosomal proteins located around the PES are uL23c (orange), uL24c (green) and uL29c (violet). Plastid‐specific features are indicated by darker colour shades.

Structural differences of the PES (marked with an asterisk) of the chloroplast 50S in comparison with the bacterial 50S subunit. The bacterial ribosomal proteins uL23 and uL24 (both in grey) are overlaid.

Adaptations of putative binding sites of cpSRP54. Corresponding residues involved in NG‐domain binding in the bacterial ribosome are shown as spheres and are labelled.

Figure 6
Figure 6. Plastid translation factor pY

Binding of plastid translation factor pY, shown in lime green, to the mRNA channel of the small subunit. The small subunit is shown from the intersubunit side. The 16S rRNA is coloured in pale yellow, and ribosomal proteins are in gold.

EM density for plastid pY. Secondary structure elements and N‐ and C‐termini are indicated.

Molecular interaction of plastid pY with 16S rRNA. The conserved nucleotides involved in A‐site decoding are coloured in red and labelled (bacterial numbering in brackets). The bacterial nucleotides A1492 and A1493 of the empty 30S subunit (PDB 1J5E; Wimberly et al, 2000) and of the 70S ribosome in complex with mRNA and tRNAs (PDB 4V51; Selmer et al, 2006) are overlaid and coloured in purple and blue, respectively. Pro127 and Arg119 of plastid pY are indicated.

Superposition of the chloroplast 70S:pY complex with bacterial A‐, P‐ and E‐site tRNAs and mRNA from the crystal structure of the Thermus thermophilus 70S ribosome (PDB 4V51; Selmer et al, 2006).

Figure EV5
Figure EV5. Plastid translation factor pY

The 70S map with density for pY in the mRNA channel is shown in grey (A) and the empty 70S map is shown in yellow (B). The 70 maps were overlaid using only the density of the 50S. Comparison of the overlaid maps, shown in 30S subunit view (C) and top view (D). The rotation of the 30S subunit is indicated with arrows.

The body and the head domains of the 30S model were independently fitted into the cryo‐EM map of the rotated state. The rotation angle of the 30S body rotation (ratcheting) (E) and the 30S head rotation (swivelling) (F) were measured using PyMOL, and the rotation axes are shown in red and blue, respectively.

Intersubunit bridges are affected by the 30S rotation. A selected residue of each intersubunit bridge is represented as white sphere in the non‐rotated state. The same residues are coloured in the rotated state either in blue, if the contact with the large subunit is maintained, or in purple, if the contact is lost. The intersubunit bridges and the differences between the rotated and the non‐rotated state are described in Appendix Table S4.

Distances (d1, d2 and d3) between backbone phosphates of selected rRNA residues (spheres) in the non‐rotated (white) and the rotated state (purple) indicating a slight opening of the mRNA channel.

tRNAs in the P/P‐ and E/E‐state from a crystal structure of the bacterial 70S ribosome in complex with mRNA and tRNAs (PDB 4V51; Selmer et al, 2006) are overlaid and shown in blue and purple, respectively. A tRNA (black) was fitted as rigid body into the 70S maps of the non‐rotated (I) and of the rotated state (J).

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