ZFIN Image: Van Haute et al., 2023, Fig. 6

Image

Figure Caption

Fig. 6

In vitro analysis of TEFM variants.

a SDS-PAGE analysis of the purified recombinant WT TEFM (amino acids 36–360) and the TEFM mutants used in the in vitro experiments. Molecular weight marker is indicated in the first lane. b DNA sequence (447–196) included in the mitochondrial Non-Coding Region (NCR), harbouring the transcription start site (+1) of the light strand promoter (LSP) and the Conserved Sequence Blocks (CSB) I, II and III. Highlighted are the CSB II sequence variants used in biochemical assays, G5AG7, G6AG7 and G8AG8. c–e CSB II sequence heterogeneity affects the capacity of TEFM mutants of preventing premature transcription termination at the CSB II region. Left panels: in vitro transcription from linearized templates that generate transcripts of ~400 nt (R.O.). Three templates with different (G)-tract sequence at CSB II were used: G5AG7 (c), G6AG7 (d) and G8AG8 (e). WT and TEFM mutants (250 fmol as a dimer) were assayed for their ability to abolish transcription termination at CSB II in each template. Right panels: The mean value of termination at different CSB II variants in the in vitro transcription. Error bars represent mean ± SD of three technical repeats, ns not significant, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 (two-tailed Student’s t test for comparison to reactions with WT TEFM). Source data are provided as a Source Data file.

Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Nat. Commun.