Departamento de Biología Experimental, Facultad de Ciencias Experimentales, Universidad de Jaén, Paraje de las Lagunillas, s/n, 23071 Jaén, Spain.
Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío, CSIC, Universidad de Sevilla, E-41013 Sevilla, Spain; Departamento de Genética, Universidad de Sevilla, E41012 Sevilla, Spain.
Biochim Biophys Acta Gene Regul Mech. 2018 Jan;1861(1):1-13. doi: 10.1016/j.bbagrm.2017.11.002. Epub 2017 Nov 11.
Rpb5 is a subunit shared by the three eukaryotic RNA polymerases although its role in transcription remains unclear. It has been proposed that it makes contact with the promoter DNA and to participate in the coordination of the opening/closing of the RNA polymerase II DNA cleft. Here, we report the specific role of Rpb5 in the function of the yeast RNA polymerase II. The rpb5-P151T mutation specifically impairs transcription elongation by RNA polymerase II but does not influence the functions of RNA polymerases I or III. The comparison of RNA polymerase II ChIP and run-on signals indicates a higher tendency to backtrack by this mutant, in agreement with its lower elongation rate and its genetic interactions with dst1Δ mutant. This phenotype is particularly striking shortly after transcription initiation and is linked to differences in the phosphorylation state of the RNA polymerase II and reduced recruitment of Spt5 to transcribe chromatin, thus influencing its anti-backtracking activity. All together, our results reveal an important role of Rpb5 in the transition from initiation to elongation mediated by the RNA polymerase II, by modulating the Spt5 association, and the backtracking activity of the enzyme.
Rpb5 是真核生物 RNA 聚合酶 III 的一个亚基，但其在转录中的作用尚不清楚。有人提出它与启动子 DNA 接触，并参与 RNA 聚合酶 II DNA 裂隙的开闭协调。在这里，我们报告了 Rpb5 在酵母 RNA 聚合酶 II 功能中的特定作用。rpb5-P151T 突变特异性地损害 RNA 聚合酶 II 的转录延伸，但不影响 RNA 聚合酶 I 或 III 的功能。RNA 聚合酶 II ChIP 和运行信号的比较表明，该突变体更容易回溯，这与其较低的延伸率以及与 dst1Δ 突变体的遗传相互作用一致。这种表型在转录起始后不久尤为明显，与 RNA 聚合酶 II 的磷酸化状态的差异以及 Spt5 募集到转录染色质的减少有关，从而影响其抗回溯活性。总之，我们的结果揭示了 Rpb5 在由 RNA 聚合酶 II 介导的从起始到延伸的转变中，通过调节 Spt5 的结合和酶的回溯活性，起着重要的作用。
