Journal: Molecular cell

Article Title: Early Scanning of Nascent Polypeptides inside the Ribosomal Tunnel by NAC.

doi: 10.1016/j.molcel.2019.06.030

Figure Lengend Snippet: Figure 3. N-bNAC Probes Nascent Chains inside the Ribosomal Tunnel (A) cryo-EM reconstruction of C. elegans NAC∙60S. (Left) Cross section of the map to better visualize the NAC density in the tunnel is shown. (Right) Close-up view of the NAC density in the tunnel that reaches the constriction formed by the extension of uL22 is shown. The resolution of the NAC density inside the ribosomal tunnel is 6–8 A˚ . The map was low-pass filtered to 4.2-A˚ resolution. The residues R126 of uL22 and R28 of eL39 shown in the stick model were substituted with Bpa in the analyses shown in (C) and Figures S5A–S5C. (B) Site-specific photo-crosslinking of different Bpa-NAC variants (see Figure S4A) to stalled ribosome-nascent chain complexes (RNCs) car- rying in vitro translated, S35-labeled NCs with defined length (10–50 aas; translated substrate = human glucose-6-phosphate isomerase [GPI]). Autoradiograph images are shown. The peptidyl- tRNA (NC-tRNA) and position-specific NC-tRNA- NAC crosslinks (arrowheads) are indicated. (C) Engineered yeast 60S ribosomes carrying Bpa at tunnel-wall-lining position R126 of FLAG-uL22, indicated in (A), were photo-crosslinked to purified human and C. elegans (C.e.) NAC. WT-NAC as well as NAC deletion mutants lacking the N-terminal aNAC domain (C. elegans DN1–53 and human DN1–67) were used. FLAG, aNAC, and bNAC im- munoblots are shown. Red arrowheads indicate bNAC-specific crosslinks. Asterisk on FLAG blot marks a NAC-independent intra-60S ribosomal crosslink. See also Figures S5A–S5C. (D) Human NAC carrying Bpa at position 2 of bNAC (b-X2) was photo-crosslinked to puromycin- washed empty 60S or untreated full 80S. eL22, uL22, and eL39 immunoblots are shown. See also Figures S4 and S5.

Article Snippet: Antibodies used throughout this study were FLAG (F7425, Sigma), uL16 (AP17603a, Abgent), eL19 (sc-100830, Santa Cruz), uL22 (14121-1-AP, Proteintech), eL22 (25002-1-AP, Proteintech), eL39 (14990-1-AP, Proteintech), uL4 (sc-100838, Santa Cruz), Gapdh (sc-137179, Santa Cruz), Actin (sc-47778, Santa Cruz), human aNAC (40-1000, Invitrogen), human bNAC (ab66940, Abcam), Puromycin (MABE343, Merck), HSP4/GRP78 (PA5-22967, Thermo Scientific), Sec61a (sc-393182, Santa Cruz), and PDI3 (kind gift of Antony Page, University of Glasgow, Scotland (Eschenlauer and Page, 2003)).

Techniques: Cryo-EM Sample Prep, In Vitro, Labeling, Autoradiography, Western Blot

Journal: Molecular cell

Article Title: Early Scanning of Nascent Polypeptides inside the Ribosomal Tunnel by NAC.

doi: 10.1016/j.molcel.2019.06.030

Figure Lengend Snippet: Figure 5. Sensing of De Novo Synthesized Nascent Chains by NAC Ribosome binding of NAC is mediated by a ribosome-binding regulatory arm (N-aNAC) and a translation sensor domain (N-bNAC). N-aNAC directly contacts the ribosome close to the tunnel exit but also possesses a ribosome binding inhibitory element that interacts with eL19. The empty tunnel of idle and early-stage translating ribosomes is sensed by N-bNAC, which inserts deeply into the ribosomal tunnel up to the constriction formed by uL22. In the tunnel-inserted conformation, NAC blocks the premature, unproductive ribosome association of Sec61 and likely of other cotranslational protein biogenesis factors, including RAC and SRP (left, early state). During polypeptide elongation, N-bNAC senses short nascent chains and is partially pushed out of the ribosomal tunnel, which likely repositions the NAC domain outside the tunnel (dotted arrows) to facilitate the early recruitment of other protein biogenesis factors, like SRP (middle, intermediate state). Once the N-bNAC tail is completely pushed out of the tunnel, it can relocate to an alternate binding site on the ribosome surface involving eL22 and eL31 (right, late stage). At this stage, NAC may orchestrate cotranslational protein folding and targeting processes by regulating the specific binding of other chaperones and targeting factors. MTS, mitochondrial targeting sequence; SS, endoplasmic reticulum signal sequence; TMD, transmembrane domain.

Article Snippet: Antibodies used throughout this study were FLAG (F7425, Sigma), uL16 (AP17603a, Abgent), eL19 (sc-100830, Santa Cruz), uL22 (14121-1-AP, Proteintech), eL22 (25002-1-AP, Proteintech), eL39 (14990-1-AP, Proteintech), uL4 (sc-100838, Santa Cruz), Gapdh (sc-137179, Santa Cruz), Actin (sc-47778, Santa Cruz), human aNAC (40-1000, Invitrogen), human bNAC (ab66940, Abcam), Puromycin (MABE343, Merck), HSP4/GRP78 (PA5-22967, Thermo Scientific), Sec61a (sc-393182, Santa Cruz), and PDI3 (kind gift of Antony Page, University of Glasgow, Scotland (Eschenlauer and Page, 2003)).

Techniques: Synthesized, Binding Assay, Sequencing