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Structure and assembly pathway of the ribosome quality control complex.

Shao S, Brown A, Santhanam B, Hegde RS - Mol. Cell (2015)

Bottom Line: During ribosome-associated quality control, stalled ribosomes are split into subunits and the 60S-housed nascent polypeptides are poly-ubiquitinated by Listerin.Structural and mutational analyses showed that ribosome-bound NEMF recruits and stabilizes Listerin's N-terminal domain, while Listerin's C-terminal RWD domain directly contacts the ribosome to position the adjacent ligase domain near the nascent polypeptide exit tunnel.Thus, highly specific nascent chain targeting by Listerin is imparted by the avidity gained from a multivalent network of context-specific individually weak interactions, highlighting a new principle of client recognition during protein quality control.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.

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Architecture of 60S-RNCs Bound to NEMF and Listerin(A) Back view of a cryo-EM reconstruction of the 60S-RNC complex showing the ribosome in gray, Listerin in orange, NEMF in teal, and the P-site peptidyl-tRNA in purple. The nascent polypeptide exit tunnel and the ribosomal L1 stalk are indicated for orientation. The map is low-pass filtered to 5 Å and displayed at a threshold to visualize continuous density of the factors.(B) View from the subunit interface of the 60S-RNC complex. The central protuberance (CP) and L1 stalk are shown for orientation. The P stalk of the ribosome is in dark gray. The N-terminal lobe (N), C-terminal lobe (C), and Middle domain (M) of NEMF are labeled.(C) Cut-away view of the 60S-RNC complex showing that the N- and C-lobes of NEMF contact the tRNA and a region of Listerin poised at the exit tunnel.(D) Example of the fit of a docked tRNA (purple), showing the 3′ CCA end, and the de novo model of the defined nascent chain (green) into the EM map density.See also Figure S3.
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fig3: Architecture of 60S-RNCs Bound to NEMF and Listerin(A) Back view of a cryo-EM reconstruction of the 60S-RNC complex showing the ribosome in gray, Listerin in orange, NEMF in teal, and the P-site peptidyl-tRNA in purple. The nascent polypeptide exit tunnel and the ribosomal L1 stalk are indicated for orientation. The map is low-pass filtered to 5 Å and displayed at a threshold to visualize continuous density of the factors.(B) View from the subunit interface of the 60S-RNC complex. The central protuberance (CP) and L1 stalk are shown for orientation. The P stalk of the ribosome is in dark gray. The N-terminal lobe (N), C-terminal lobe (C), and Middle domain (M) of NEMF are labeled.(C) Cut-away view of the 60S-RNC complex showing that the N- and C-lobes of NEMF contact the tRNA and a region of Listerin poised at the exit tunnel.(D) Example of the fit of a docked tRNA (purple), showing the 3′ CCA end, and the de novo model of the defined nascent chain (green) into the EM map density.See also Figure S3.

Mentions: The combined data set containing 117,461 particles was processed through RELION (Scheres, 2012). After initial 3D refinement, movie processing was performed to adjust for drift and radiation damage (Bai et al., 2013), resulting in an initial map that showed extraribosomal density we provisionally assigned to Listerin and NEMF. These particles were then subjected to further 3D classification with a mask around the presumed Listerin and NEMF densities to enrich for their occupancy. The enriched class, containing 63,826 particles, was refined (Table 1) to produce our final map of a 60S-RNC in complex with Listerin and NEMF (Figure 3). Gold standard FSC curve analysis indicated an overall resolution of 3.6 Å (Figure S3C). The ribosome displayed the highest resolution, while local resolution for the associated factors decreased relative to the core of the ribosome (Figure S3D).


Structure and assembly pathway of the ribosome quality control complex.

Shao S, Brown A, Santhanam B, Hegde RS - Mol. Cell (2015)

Architecture of 60S-RNCs Bound to NEMF and Listerin(A) Back view of a cryo-EM reconstruction of the 60S-RNC complex showing the ribosome in gray, Listerin in orange, NEMF in teal, and the P-site peptidyl-tRNA in purple. The nascent polypeptide exit tunnel and the ribosomal L1 stalk are indicated for orientation. The map is low-pass filtered to 5 Å and displayed at a threshold to visualize continuous density of the factors.(B) View from the subunit interface of the 60S-RNC complex. The central protuberance (CP) and L1 stalk are shown for orientation. The P stalk of the ribosome is in dark gray. The N-terminal lobe (N), C-terminal lobe (C), and Middle domain (M) of NEMF are labeled.(C) Cut-away view of the 60S-RNC complex showing that the N- and C-lobes of NEMF contact the tRNA and a region of Listerin poised at the exit tunnel.(D) Example of the fit of a docked tRNA (purple), showing the 3′ CCA end, and the de novo model of the defined nascent chain (green) into the EM map density.See also Figure S3.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321881&req=5

fig3: Architecture of 60S-RNCs Bound to NEMF and Listerin(A) Back view of a cryo-EM reconstruction of the 60S-RNC complex showing the ribosome in gray, Listerin in orange, NEMF in teal, and the P-site peptidyl-tRNA in purple. The nascent polypeptide exit tunnel and the ribosomal L1 stalk are indicated for orientation. The map is low-pass filtered to 5 Å and displayed at a threshold to visualize continuous density of the factors.(B) View from the subunit interface of the 60S-RNC complex. The central protuberance (CP) and L1 stalk are shown for orientation. The P stalk of the ribosome is in dark gray. The N-terminal lobe (N), C-terminal lobe (C), and Middle domain (M) of NEMF are labeled.(C) Cut-away view of the 60S-RNC complex showing that the N- and C-lobes of NEMF contact the tRNA and a region of Listerin poised at the exit tunnel.(D) Example of the fit of a docked tRNA (purple), showing the 3′ CCA end, and the de novo model of the defined nascent chain (green) into the EM map density.See also Figure S3.
Mentions: The combined data set containing 117,461 particles was processed through RELION (Scheres, 2012). After initial 3D refinement, movie processing was performed to adjust for drift and radiation damage (Bai et al., 2013), resulting in an initial map that showed extraribosomal density we provisionally assigned to Listerin and NEMF. These particles were then subjected to further 3D classification with a mask around the presumed Listerin and NEMF densities to enrich for their occupancy. The enriched class, containing 63,826 particles, was refined (Table 1) to produce our final map of a 60S-RNC in complex with Listerin and NEMF (Figure 3). Gold standard FSC curve analysis indicated an overall resolution of 3.6 Å (Figure S3C). The ribosome displayed the highest resolution, while local resolution for the associated factors decreased relative to the core of the ribosome (Figure S3D).

Bottom Line: During ribosome-associated quality control, stalled ribosomes are split into subunits and the 60S-housed nascent polypeptides are poly-ubiquitinated by Listerin.Structural and mutational analyses showed that ribosome-bound NEMF recruits and stabilizes Listerin's N-terminal domain, while Listerin's C-terminal RWD domain directly contacts the ribosome to position the adjacent ligase domain near the nascent polypeptide exit tunnel.Thus, highly specific nascent chain targeting by Listerin is imparted by the avidity gained from a multivalent network of context-specific individually weak interactions, highlighting a new principle of client recognition during protein quality control.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.

Show MeSH
Related in: MedlinePlus