Extended Data Fig. 1: Efficiency of Np4A incorporation during transcription initiation.
From: Molecular basis for noncanonical transcription initiation from Np4A alarmones
a-c, Determination of the relative efficiencies of transcription initiation with Ap4A vs. ATP in the first transcription step. a, Representative gel showing the initial RNA products of transcription reactions performed in the presence of 0.75 mM Ap4A and 0.008-1.8 mM ATP as the initiating nucleotides and [α32P]-CTP as the extending nucleotide. The experiment was repeated 3 times with similar results, with quantification shown in panels (b) and (c). b, Graph showing the fraction of Ap4A-initiated RNA vs. the Ap4A/ATP ratio in transcription reactions conducted with tDNAs containing various -1 nucleotides. The curves are logarithmic regression fits using the three highest concentrations of ATP (0.52, 0.90, 1.80 mM) from panel (a). Each point is the mean ± SD of three independent experiments (n = 3). c, Relative efficiencies of transcription initiation with Ap4A vs. ATP [(kcat/Km, Ap4A)/(kcat/Km, ATP)] determined from panel (b). Each bar is the mean ± SD of three independent experiments (n = 3). d, Promoter sequences used to validate that DNA templates for cryo-EM studies of Tt-RNAP show expected differences in transcription efficiency from Np4A with purines or pyrimidines at the -1 position of tDNA. Nucleotides in orange differ from the promoter in Fig. 1e. e-f, Efficiency of Np4A incorporation by Tt-RNAP from the promoter described in panel (d). The efficiency was determined as in Fig. 1. e, A representative gel used for quantification. f, A bar graph showing data points from two independent experiments (n = 2).
