Fig. 4: Decoding of AAA and m6AAA monitored by smFRET.
From: Modulation of translational decoding by m6A modification of mRNA
a Schematic of the smFRET experiment. 70S IC (light gray) is labeled with Cy3 (green star) at the protein L11 and Lys-tRNALys is labeled with Cy5 (red star). b Relative abundance of the different types of traces during decoding of AAA (black) or m6AAA (gray) codon. “Rejected” denotes the fraction of complexes that dissociate from the ribosome after initial binding (IB; c, d). “Decoding” pertains to traces that show the characteristic codon reading (CR) phase followed by accommodation and ensuing fluctuations between A/A, A/P, and A/P* states (e, f). “Post-decoding” are the complexes in which Lys-tRNALys is found in an A/A, A/P or A/P* state at the start of the recording (g, h). “Stalled” denotes the fraction of ribosomes that do not progress past the CR state (Supplementary Fig. 2). Shown are the mean values with error bars representing standard deviation from N = 3 independent experiments. ** indicates statistical significance with P < 0.01 using a two-tailed Welch’s t-test without adjustment for multiple comparisons. P = 0.0015 for rejected traces, P = 0.0038 for post-decoding traces, P = 0.0565 for decoding traces. Open circles are the values in the individual experiments. c Contour plot showing the distribution of FRET values after synchronization during initial binding and dissociation of Lys-tRNALys(Cy5) from ICs carrying the AAA codon in the A site. Traces are synchronized to the first transition with FRET > 0. N, number of traces. d same as c on the m6AAA codon. e Contour plot showing the distribution of FRET values during the full decoding trajectory on the AAA codon. FRET 0.96 ± 0.03 in the CR state derived from mean ± s.d. of the µ values of the Gaussian fitting of N = 3 independent experiments. f same as e on the m6AAA codon, FRET 0.98 ± 0.01. g Contour plot showing the distribution of FRET values in the post-decoding complexes carrying fMet-Lys-tRNALys(Cy5) on the AAA codon. FRET population distribution reveals two states with FRET 0.79 ± 0.01 (A/A, A/P) and 0.58 ± 0.02 (A/P*)35,64,65,66. h Same as g on m6AAA codon. A/A, A/P, FRET 0.80 ± 0.01; A/P*, FRET 0.61 ± 0.01. Data are from N = 3 independent experiments. Source data are provided as a Source Data file.
