in vitro tnttm quick coupled transcription/translation system  (Promega)

Journal: bioRxiv

Article Title: Sequence-specific trapping of EF-Tu/glycyl-tRNA complex on the ribosome by bottromycin

doi: 10.1101/2025.08.17.670399

Figure Lengend Snippet: ( a ) Overview of the structure of the E. coli 70S ribosome stalled by BOT (yellow) during in vitro translation of the dapG mRNA. The 30S and 50S subunits are shown in light yellow and light blue, respectively; EF-Tu is in teal; the mRNA is in blue, and the A-and P-site tRNAs are in dark blue and orange, respectively. BOT traps glycyl-tRNA in complex with EF-Tu on the ribosome in an A/T-state, preventing proper accommodation into the A site. The position of the fully accommodated A/A tRNA is indicated by a black outline. ( b ) Close-up view of the ribosome-bound EF-Tu•GDP•Gly-tRNA ternary complex with BOT bound at the interface between EF-Tu domains I and II (teal and green), adjacent to the CCA-end of the Gly-tRNA (dark blue). GDP (orange) in the GTPase center indicates a post-hydrolysis state of EF-Tu. ( c ) Cryo-EM density map (blue mesh) for BOT with the refined atomic model overlaid. Two orthogonal views reveal well-resolved density for all key chemical moieties of the drug, allowing confident placement and modeling. ( d, e ) Detailed views of the interaction network between BOT, EF-Tu, and the CCA-end of Gly-tRNA. BOT forms an extensive H-bonding interface with EF-Tu domain I (Asn64-Val68), domain II (Asp217 and Arg263) residues, and the phosphate of nucleotide C75. H-bonds are shown as black dotted lines. ( f ) CH-π stacking between the β-methyl-phenylalanine (mPhe) side chain of BOT and the glycine moiety of the aa-tRNA. This highly specific interaction explains the strict selectivity of BOT for glycyl-tRNA and its amino acid-dependent inhibitory activity.

Article Snippet: Toeprinting analysis was carried out using model mRNA templates listed in and PURExpress E. coli in vitro transcription-translation coupled system (NEB) as described previously .

Techniques: In Vitro, Cryo-EM Sample Prep, Activity Assay

Journal: bioRxiv

Article Title: Sequence-specific trapping of EF-Tu/glycyl-tRNA complex on the ribosome by bottromycin

doi: 10.1101/2025.08.17.670399

Figure Lengend Snippet: ( a ) Characteristics of B. subtilis mutants selected on BOT-containing agar plates. All resistant clones carried point mutations at Asp218 residue in the tuf gene. ( b ) Location of residue Asp218 ( B. subtilis numbering; equivalent to Asp217 in E. coli ) within EF-Tu, mapped onto its domain organization. ( c, d ) In silico modeling of the D217Y ( E. coli numbering) substitution in the structure of EF-Tu/Gly-tRNA/BOT complex reveals a steric clash between the bulky tyrosine side chain and the bound BOT molecule. ( e ) Schematic of engineered B. subtilis strains harboring a second copy of the tuf gene inserted at the chromosomal lacA locus under the control of an xylose-inducible promoter. ( f ) Heat map showing the growth (OD 600 ) of the four strains shown in ( e ) in SM minimal medium supplemented with increasing concentrations of BOT and 1% xylose. ( g ) Growth of the same strains on SM minimal agar plates supplemented with 1% xylose, without the drug (top) or with 2 µg/mL BOT (bottom). ( h ) Distribution of tuf gene paralogs (encoding EF-Tu) across complete bacterial genomes from major classes/phyla. Selected species known to be highly sensitive to BOT are listed in the insets along with their respective tuf copy number.

Article Snippet: Toeprinting analysis was carried out using model mRNA templates listed in and PURExpress E. coli in vitro transcription-translation coupled system (NEB) as described previously .

Techniques: Clone Assay, Residue, In Silico, Control