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. 2022 Jul 22;13:4231. doi: 10.1038/s41467-022-31852-w

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Schematic of translocation on slippery mRNA with EF-G(wt). Pept-tRNA^Lys-BHQ2 (black circle) moves from the A to the P site. The near-cognate –1-frame Val-tRNA^Val-Cy5 (red star) samples POST2 complexes without accommodating in the A site. EF-G is added to immobilized PRE1 complexes together with the EF-Tu–GTP–Val-tRNA^Val-Cy5 complex. b Representative time trace of pept-tRNA^Lys-BHQ2 movement and subsequent sampling of POST2 complexes by Val-tRNA^Val-Cy5 (N = 89). Fl, fluorescence intensity; Q-pb, photobleaching of BHQ2. Green labels at the right Y-axis indicate tRNA^Lys conformational states monitored by Cy3 fluorescence, as assigned in Supplementary Fig. 4. c Zoom-in into b showing Cy3 and Cy5 FI and calculated FRET of the initial binding (IB) without accommodation of –1-frame Val-tRNA^Val-Cy5 on POST2 complexes. d Contour plot showing distribution of FRET values during –1-frame Val-tRNA^Val-Cy5 sampling of POST2 complexes. Traces were synchronized to the point with FRET > 0. Histogram at the right shows FRET distribution after synchronization. e Schematic of translocation on slippery mRNA by EF-G(wt), with −1 frameshifting. After translocation of pept-tRNA^Lys-BHQ2 (black circle) from the A to the P site and Val-tRNA^Val-Cy5 (red star) can bind to its cognate –1-frame codon. f Representative time trace of pept-tRNA^Lys-BHQ2 translocation and accommodation of Val-tRNA^Val-Cy5 (N = 57). g Zoom-in into f showing Cy3 and Cy5 FI and calculated FRET of Val-tRNA^Val-Cy5 binding to POST2 forming PRE2 complexes. Red labels indicate conformational states of tRNA^Val-Cy5. h Contour plot showing distribution of FRET values after Val-tRNA^Val-Cy5 accommodation on POST2 complex forming PRE2. i Schematic of translocation on slippery mRNA by EF-G(Q507D), with −1 frameshifting. After translocation of pept-tRNA^Lys-BHQ2 from the A to the P site, Val-tRNA^Val-Cy5 (red star) accommodates on its cognate codon in –1-frame. j Representative time trace of pept-tRNA^Lys-BHQ2 translocation and subsequent accommodation of –1-frame Val-tRNA^Val-Cy5 (box) (N = 46). k Zoom-in into k showing Cy3 and Cy5 FI and calculated FRET of Val-tRNA^Val-Cy5 binding to POST2 complex, leading to CR and subsequent fluctuations between A/A, A/P and A/P* states. l Contour plot showing the distribution of FRET values after Val-tRNA^Val-Cy5 accommodation on POST2 complex.