Journal: bioRxiv

Article Title: The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis

doi: 10.1101/2025.05.27.656346

Figure Lengend Snippet: β-gal structure. Top, organisation of domains 1-5 (D1-5). Left, monomer structure (PDB: 6CVM ). Right, tetramer structure showing long and activating interfaces. b, Refolding of β-gal upon dilution from 8 M urea into buffer at 10 or 37 °C, assayed by recovery of enzyme activity. Data were fit to a single exponential function. Yield = 25.1%; 95% CI 24.5-25.7%. Half-time = 4.3 h; 95% CI 3.9-4.6 h. Error bars represent s.d. (n = 3). c, Specific activity of β-gal purified from E. coli (n=8 technical replicates) or produced by in vitro translation (IVT) (n=3 independent IVT reactions). Data were normalised to the mean specific activity of purified β-gal. Error bars represent s.d. d, Amount and activity of in vitro translated full-length β-gal, at different times after initiating IVTs using a plasmid encoding β-gal. Error bars represent s.d. (n = 3 independent IVT reactions). e, Accumulation of β-gal activity during IVT reaction. After 40 min (arrow), reactions were treated with 5 mM chloramphenicol (CAM), 5 mM puromycin (PURO), or an equal volume of water (H 2 O). f, β-gal activity in ribosomal fraction of IVTs. β-gal was expressed for 1 h before supplementing the reaction with either CAM to stabilise ribosome-nascent chain complexes, or PURO to release nascent chains. The ribosomal fraction was isolated by sucrose cushion ultracentrifugation. Error bars represent s.d. (n = 3 independent IVT reactions). ns - p>0.05; * - p<0.05 based on one-way ANOVA with Dunnett’s multiple comparisons. g, Oligomeric state of natively folded (native), refolded, or IVT-produced β-gal was analysed using native-PAGE. Duplicate gels were immunoblotted for β-gal or stained with the β-gal substrate X-gal. Bands corresponding to the tetramer (T), dimer (D), monomer (M) or intermediate (I) are indicated.

Article Snippet: BL21(DE3) E. coli ΔlacZ cells (Addgene, 99247 ) transformed with plasmid encoding C-terminally truncated β-gal 1-440, 1-519, 1-725 or β-gal domain 5 (D5, 739-1023) were grown in LB at 37 °C until OD 600 0.6–0.8 and protein expression was subsequently induced with 1 mM IPTG for an additional 18 hours at 16-18 °C.

Techniques: Activity Assay, Purification, Produced, In Vitro, Plasmid Preparation, Isolation, Clear Native PAGE, Staining

Journal: bioRxiv

Article Title: The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis

doi: 10.1101/2025.05.27.656346

Figure Lengend Snippet: β-gal refolding upon dilution from 8 M urea into buffer at different temperatures, assayed by recovery of enzyme activity. Data were fit to a single exponential function. Error bars represent s.d. (n = 3). b, Arrhenius plot showing temperature dependence of β-gal refolding kinetics as measured in ( a ). E a = 84±1 kJ.mol -1 . Error bars represent s.d. (n = 3). c, Influence of Hsp70 chaperone system on β-gal refolding. Refolding was measured as in (a), at 37 °C. The reaction was supplemented with purified DnaK, DnaJ and GrpE, with or without ATP. Data were fit to a single exponential function, giving t ½ = 11 min (95% CI, 8 – 14 min) in the presence of ATP. Error bars represent s.d. (n = 3). d, Aggregation during β-gal refolding. Turbidity was monitored by absorbance at 320 nm. Refolding under optimal conditions as in (10 °C, final urea concentration of 1.5 M) did not result in detectable turbidity. Turbidity could be induced by raising the temperature of the reaction to 25 °C and reducing the final urea concentration to 0.16 M). The shaded region represents s.d. (n = 3). e , Oligomeric state of β-gal during refolding, analysed using native-PAGE. Replicate gels were stained with Coomassie, immunoblotted for β-gal, or stained with the β-gal substrate X-gal. Bands corresponding to the tetramer (T), dimer (D), monomer (M) or intermediate (I) are indicated. Natively folded β-gal purified from E.coli is included as a control.

Article Snippet: BL21(DE3) E. coli ΔlacZ cells (Addgene, 99247 ) transformed with plasmid encoding C-terminally truncated β-gal 1-440, 1-519, 1-725 or β-gal domain 5 (D5, 739-1023) were grown in LB at 37 °C until OD 600 0.6–0.8 and protein expression was subsequently induced with 1 mM IPTG for an additional 18 hours at 16-18 °C.

Techniques: Activity Assay, Purification, Concentration Assay, Clear Native PAGE, Staining, Control

Journal: bioRxiv

Article Title: The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis

doi: 10.1101/2025.05.27.656346

Figure Lengend Snippet: Coomassie-stained gel of RNCs purified from E.coli lacking endogenous Trigger factor (Δ tig ) or β-gal (Δ lacZ ). Ribosomal proteins (r.p.), Trigger factor (TF), the nascent chain linked to its peptidyl-tRNA (*) and co-purifying full-length β-gal (**) are indicated. Purified β-gal and empty 70S ribosomes are shown alongside for reference.

Article Snippet: BL21(DE3) E. coli ΔlacZ cells (Addgene, 99247 ) transformed with plasmid encoding C-terminally truncated β-gal 1-440, 1-519, 1-725 or β-gal domain 5 (D5, 739-1023) were grown in LB at 37 °C until OD 600 0.6–0.8 and protein expression was subsequently induced with 1 mM IPTG for an additional 18 hours at 16-18 °C.

Techniques: Staining, Purification

Journal: bioRxiv

Article Title: The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis

doi: 10.1101/2025.05.27.656346

Figure Lengend Snippet: β-gal enzyme activity of 5 nM RNCs purified from wild-type E. coli . Error bars represent s.d. (n = 3). b, Endogenous β-gal assembles with RNCs. Enzyme activity of 5 nM RNCs purified from wild-type, or Δ lacZ E. coli which do not express β-gal. Error bars represent s.d. (n = 3). c, Onset of co-post β-gal assembly in vivo. SeRP data showing the codon-resolved enrichment (selected translatome / total translatome) of β-gal-bound RNCs along the lacZ open reading frame. d, D1-4 are active off but not on the ribosome. Enzyme activity of 120 nM 1-725 β-gal truncation (domains 1-4) and RNC 1-745 , both purified from Δ lacZ E. coli . Error bars represent s.d. (n = 3). e, Truncated β-gal misassembles. Negative-stain EM micrograph of β-gal 1-725 purified from Δ lacZ E. coli. Particles corresponding to tetramers or higher-order oligomers are indicated with blue or white circles, respectively. f, β-gal assembly is triggered by folded D5. Enzyme activity of 7.5 nM β-gal RNCs purified from Δ lacZ E. coli , in the absence or presence of isolated folded domain 5 (D5, residues 739-1023). The distance between D1-4 (residues 1-725) and the ribosome was increased by inserting a 50 or 100 aa Gly-Ser linker between the C-terminus of the NC and the stalling sequence. g, D5 folds primarily post-translation. Difference in deuterium uptake, after 100 s deuteration, between native β-gal and RNC 1-1014 purified from Δ lacZ E. coli . Regions that are deprotected in the RNC are coloured in shades of red. The C-terminal 30 aa, expected to be inside the exit tunnel in RNC 1-1014 , are coloured green.

Article Snippet: BL21(DE3) E. coli ΔlacZ cells (Addgene, 99247 ) transformed with plasmid encoding C-terminally truncated β-gal 1-440, 1-519, 1-725 or β-gal domain 5 (D5, 739-1023) were grown in LB at 37 °C until OD 600 0.6–0.8 and protein expression was subsequently induced with 1 mM IPTG for an additional 18 hours at 16-18 °C.

Techniques: Activity Assay, Purification, In Vivo, Staining, Isolation, Sequencing

Journal: bioRxiv

Article Title: The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis

doi: 10.1101/2025.05.27.656346

Figure Lengend Snippet: Isolated RNCs are inactive. Stalling constructs were expressed in PURE IVT reactions as in . Enzyme activity was monitored in real-time by fluorescence upon cleavage of the substrate resorufin-β-D-glucopyranoside. Full-length β-gal (with a stop codon) was translated as a control. b , Copurification of endogenous full-length β-gal with RNC 1-1014 . Wild-type RNC 1-1014 (RNC 1-1014WT ) or a variant with a disrupted activation interface (RNC 1-1014Δ11-41 ) were purified from wild-type (WT), Δ lacZ or Δ tig E. coli and resolved by SDS-PAGE followed by Coomassie staining. The last lane contains purified full-length β-gal for reference. Bands corresponding to the NCs (*), copurifying full-length (**) or C-terminally truncated (***) β-gal, Trigger factor (TF) and ribosomal proteins (r.p.) are indicated. Right: immunoblot of RNC 1-1014WT purified from wild-type cells, probed using an antibody against the β-gal N-terminus (ab106567). c , Copurifying full-length β-gal is protease-resistant compared to the NC. RNC 1-1014 purified from WT or Δ lacZ cells was treated with Proteinase K and resolved by SDS-PAGE followed by Coomassie staining. Bands corresponding to the NC (*) and to the full-length β-galactosidase (**) are indicated. d, β-gal pulldown specifically enriches lacZ mRNA. Comparison of the gene-specific footprint abundance in the β-galactosidase co-IP sample and the total translatome in RPKM (reads per kilobase per million mapped reads). The lacZ transcript is indicated in blue. e , 1-519 β-gal truncation is monomeric. Molecular weight was measured by SEC-MALS at different concentrations as indicated. Molecular weight (MW, left y-axis) and ultraviolet absorbance (UV, right y-axis) are plotted as a function of elution time. The theoretical mass of the 1-519 fragment is 59 kDa. f , lacZ -translating ribosomes are not enriched in the disome fraction. Comparison of the gene-specific footprint abundance in disome and monosome samples in RPKM (reads per kilobase per million mapped reads). The lacZ transcript is indicated in yellow. g, β-gal does not assemble via a co-co mechanism. Monosome (blue) and disome (yellow) footprint density along the lacZ open reading frame. RPM: reads per million. h , 1-725 β-gal truncation is partially tetrameric. SEC-MALS was performed as in ( d ), at a concentration of 30 μM. Two species of different molecular weights were detected, corresponding to monomeric (87 kDa) and tetrameric (320 kDa) forms. The theoretical mass of the 1-725 fragment is 83 kDa. i , Full-length β-gal forms tetramers rather than higher order assemblies as detected in for the 1-725 fragment. Negative stain electron microscopy micrograph of purified full-length β-gal. The scale bar corresponds to 50 nm.

Article Snippet: BL21(DE3) E. coli ΔlacZ cells (Addgene, 99247 ) transformed with plasmid encoding C-terminally truncated β-gal 1-440, 1-519, 1-725 or β-gal domain 5 (D5, 739-1023) were grown in LB at 37 °C until OD 600 0.6–0.8 and protein expression was subsequently induced with 1 mM IPTG for an additional 18 hours at 16-18 °C.

Techniques: Isolation, Construct, Activity Assay, Fluorescence, Control, Copurification, Variant Assay, Activation Assay, Purification, SDS Page, Staining, Western Blot, Comparison, Co-Immunoprecipitation Assay, Molecular Weight, Concentration Assay, Electron Microscopy