Transcriptional regulation

Stuck in traffic

Mol. Cell 63, 433–444 (2016)

Kin28 is a kinase within the yeast TFIIH complex. Some evidence indicates a role for Kin28 in regulating transcription initiation and elongation through phosphorylation of the C-terminal domain of RNA polymerase II (Pol II), but reversible Kin28 inhibitor studies revealed minor effects on cellular viability and mRNA synthesis, suggesting that a low level of Kin28 activity may be sufficient for transcriptional initiation. Rodriguez-Molina et al. proposed that irreversible/covalent inhibition might reduce Kin28 activity levels below this threshold and used a chemical-genetic approach to engineer mutations in the Kin28 ATP-binding pocket to enable binding by an ATP analog containing a thiol-reactive chloromethyl-ketone moiety (CMK). Yeast strains expressing this Kin28 irreversible-inhibitor-sensitized allele (kin28is) with CMK exhibited cellular lethality and greatly reduced serine phosphorylation in the C-terminal domain, resembling KIN28 genetic mutants. Transcriptome analysis of kin28is with CMK revealed low levels of nascent mRNA, and Pol II tracking studies exhibited a buildup of Pol II at 5′ ends of protein-coding genes, revealing a role for Kin28 in promoting transcriptional elongation. Consistent with a defect in elongation activity, combining kin28is with a fast-processing RNA polymerase variant rescued the growth defects. Altogether, these studies present a structure-guided approach to irreversibly sensitizing a desired kinase and underscore the power of the covalent inactivation strategy to resolve biological questions.

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Miura, G. Stuck in traffic. Nat Chem Biol 12, 763 (2016). https://doi.org/10.1038/nchembio.2197

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