1. Dynamic transcriptome analysis in yeast.
Dynamic transcriptome analysis in yeast. (A) Scheme of metabolic mRNA labeling in yeast. Nascent mRNA is labeled with 4‐thiouridine (4sU) and thiol‐specifically biotinylated following cell lysis and preparation of total cellular RNA. Fractionation into pre‐existing and nascent mRNA is achieved with streptavidin beads. (B) The yeast transcriptome is undisturbed by expression of the human nucleoside transporter hENT1. The volcano plot compares mRNA levels after 6 min labeling versus wild‐type cells without labeling. Each dot corresponds to one gene, the x axis displays the log2(fold) of that gene, the y axis represents the multiple testing adjusted P‐value (see Supplementary Section 12.5). In all, 17 genes showed a significant change in mRNA levels (adjusted P‐value <5%), only three of which were at least twofold. The results for the other tested labeling periods are given in the Supplementary Section (C) 4sUTP is modeled into the crystal structure of a Pol II transcribing complex (PDB code: 116H). The thiol group at position 4 can form a hydrogen bond with the DNA template strand (blue). Nascent RNA is in red.
Christian Miller et al. Mol Syst Biol 2011;7:458
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