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. 2015 Jan 2;347(6217):75-8.
doi: 10.1126/science.1259724.

Protein Synthesis. Rqc2p and 60S Ribosomal Subunits Mediate mRNA-independent Elongation of Nascent Chains

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Free PMC article

Protein Synthesis. Rqc2p and 60S Ribosomal Subunits Mediate mRNA-independent Elongation of Nascent Chains

Peter S Shen et al. Science. .
Free PMC article

Abstract

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein--not an mRNA--determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails").

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. CryoEM reconstructions of peptidyl-tRNA-60S ribosomes bound by the RQC components Rqc2p and Ltn1p
A) A peptidyl-tRNA-60S complex isolated by immunoprecipitation of Rqc1p. The ribosome density is transparent to visualize the nascent chain. B) Rqc2p (purple) and an ~A-site tRNA (yellow) bound to peptidyl-tRNA-60S complexes. Landmarks indicated (L1, L1 stalk; SB, P-stalk base). C) Ltn1p (tan) bound to Rqc2p-peptidyl-tRNA-60S complexes (B).
Figure 2
Figure 2. Rqc2p binding to the 60S ribosome, ~P-site and ~A-site tRNAs
A) Rqc2p contacts ~P- and ~A-site tRNAs, the sarcin-ricin loop (SRL) and P-stalk base rRNA (SB). B) Rigid body fitting of tRNAs structures (ribbons) into EM densities (mesh).
Figure 3
Figure 3. Rqc2p-dependent enrichment of tRNAAla(IGC) and tRNAThr(IGU)
A) tRNA cDNA reads extracted from purified RQC particles and summed per unique anticodon, with versus without Rqc2p. B) Secondary structures of tRNAAla(IGC) and tRNAThr(IGU). Identical nucleotides underlined. Edited nucleotides indicated with asterisks (24, 25). C) Weblogo representation of cDNA sequencing reads related to shared sequences found in anticodon loops (positions 32-38) of mature tRNAAla(IGC) and tRNAThr(IGU)(26). D) ~A-tRNA contacts with Rqc2p at the D-, T-, and anticodon loops. Identical nucleotides between tRNAAla(IGC) and tRNAThr(IGU) colored as in panel B (A, green; U, red; C, blue; G, orange) and pyrimidine, purple. Anticodon nucleotides are indicated as slabs.
Figure 4
Figure 4. Rqc2p-dependent formation of CAT tails
A–B, D) Immunoblots of stalling reporters in RQC deletion strains. C) Total amino acid analysis of immunoprecipitated GFP expressed in ltn1Δ and ltn1Δrqc2Δ strains, N=3. E) Triplicate GFP levels measured with a flow cytometer and normalized to a wild type control. EV=empty vector. All error bars are standard deviations.

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