Fig. 6. Binding of leaderless mt-mRNA to the monosome.
a An optical tweezers setup to study mitochondrial translation initiation. RNA construct containing the first 51 nucleotides of MT-CO2 mRNA was ligated to biotinylated lambda DNA, allowing the 5′-end accessible for mitoribosome binding. b In order to monitor both mitoribosomal subunits, mtLSU and mtSSU were labeled with Cy5 and Atto488, respectively, and initiation reactions were made by incubating either double-labeled monosome (b, 1.) or Cy5-labeled mtSSU (b, 3.) with the DNA/RNA hybrid, mtIF3, mtIF2 (GTP), and fMet-tRNAMeti. An RNA construct without start codon was used as a negative control (b, 2.). c Representative images showing binding of the mitoribosomal subunits tested by simultaneous dual-color confocal imaging. d Quantification of the binding events. The number of detected fluorescently labeled species were normalized to the number of trapped imaged DNA. Bars represent mean ± SD. Source data are provided as a Source Data file.