Extended Data Figure 3. S4 labeling and its binding to the rRNA.

E. coli ribosomal protein S4 was over-expressed, purified, and labeled with Cy3 or Cy5 fluorescent dyes as described in Methods. a, SDS-PAGE of unlabeled protein stained with Coomassie (left) or labeled with Cy5 (right). b, c, Ensemble titration of the modified 5′ domain RNAs in HKM20 shows that S4-Cy5 binds with similar affinity as the wild type S4-rRNA complex. Extended 5′ domain RNAs annealed with h3P5-Cy3 and/or h16 oligonucleotides were titrated with S4-Cy5 in a 500 μL cuvette, and the fluorescence emission was recorded from 550 to 700 nm with 540 nm excitation (Fluorolog-3, Horiba). Excitation and emission slits were fixed at 2 nm and 5 nm, respectively. The sample was incubated at 37 ºC for 1 min after each addition. Two or more independent measurements were averaged and titration curves were fitted to a quadratic binding expression. Equilibrium dissociation constants were 5′dom-h3, 0.11 ± 0.02 nM (statistical error of the fit parameter) and 5′dom-h3h16, 0.2 ± 0.1 nM, at 37 ºC, and were comparable to that of the 5′ domain RNA with wild-type E. coli S4 (0.9 nM)¹⁷.