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. 2024 Oct 17;7:1345. doi: 10.1038/s42003-024-07008-5

Fig. 2. The tbpA riboswitch modulates translation initiation through cotranscriptional TPP sensing.

Fig. 2

a Schematics representing the toeprint assays. Left, in the absence of TPP, the formation of the ON state allows the binding of the 30S ribosomal subunit. In such a case, the reverse transcriptase (RT) is expected to stop elongating upon reaching the bound 30S subunit. The Shine-Dalgarno (SD) and the GUG start codon are shown in blue and red, respectively. The anti-P1 stem is shown in orange. The DNA oligo used to produce the cDNA is shown in gray. Right, in the presence of TPP, the formation of the OFF state prevents the binding of the 30S subunit, thereby not producing a toeprint at the ribosome binding site. The P1 stem is shown in green. b Left, toeprint assays performed on the tbpA transcript as a function of the 30S subunit, tRNA-fMet and TPP. The toeprint at position C144 is preferentially obtained with the 30S subunit and tRNA-fMet, but is decreased upon TPP binding. Right, quantification of the toeprint efficiency. The experiments have been performed three times and the average and the standard deviations are shown. c Experimental assays monitoring TPP binding using in vitro transcription-translation assays. In these assays, transcription is first allowed to proceed until the addition of rifampicin. The second step involves the addition of amino acids to permit translation. The effect of TPP on the translation efficiency is assessed by adding it during the first (cotranscriptional) or second (post-transcriptional) step. d Transcription-translation assays assessing TPP binding to the riboswitch. Reactions were achieved in the absence (-) or presence of TPP added either cotranscriptionally (Co-trx) or post-transcriptionally (Post-trx) as indicated in c. The ratio of repression is shown below the gels. e Control experiments for transcription-translation assays were performed with lacZ. No translation repression was obtained when adding TPP cotranscriptionally. The ratio of repression is indicated below the gels.