Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0024, USA.
J Biol Chem. 2011 May 27;286(21):18825-33. doi: 10.1074/jbc.M110.202119. Epub 2011 Apr 5.
Spt5 is a transcription factor conserved in all three domains of life. Spt5 homologues from bacteria and archaea bind the largest subunit of their respective RNA polymerases. Here we demonstrate that Spt5 directly associates with RNA polymerase (Pol) I and RNA Pol II in yeast through its central region containing conserved NusG N-terminal homology and KOW domains. Deletion analysis of SPT5 supports our biochemical data, demonstrating the importance of the KOW domains in Spt5 function. Far Western blot analysis implicates A190 of Pol I as well as Rpb1 of Pol II in binding Spt5. Three additional subunits of Pol I may also participate in this interaction. One of these subunits, A49, has known roles in transcription elongation by Pol I. Interestingly, spt5 truncation mutations suppress the cold-sensitive phenotype of rpa49Δ strain, which lacks the A49 subunit in the Pol I complex. Finally, we observed that Spt5 directly binds to an essential Pol I transcription initiation factor, Rrn3, and to the ribosomal RNA. Based on these data, we propose a model in which Spt5 is recruited to the rDNA early in transcription and propose that it plays an important role in ribosomal RNA synthesis through direct binding to the Pol I complex.
Spt5 是一种在所有三个生命领域中都保守的转录因子。细菌和古菌的 Spt5 同源物与各自 RNA 聚合酶的大亚基结合。在这里，我们通过其包含保守的 NusG N 端同源和 KOW 结构域的中心区域证明，Spt5 直接与酵母中的 RNA 聚合酶（Pol）I 和 RNA Pol II 相关联。SPT5 的缺失分析支持我们的生化数据，表明 KOW 结构域在 Spt5 功能中的重要性。远 Western blot 分析表明，Pol I 的 A190 以及 Pol II 的 Rpb1 参与与 Spt5 的结合。Pol I 的另外三个亚基也可能参与这种相互作用。其中一个亚基 A49 在 Pol I 的转录延伸中具有已知的作用。有趣的是，spt5 截断突变抑制了 rpa49Δ 菌株的冷敏感表型，该菌株缺乏 Pol I 复合物中的 A49 亚基。最后，我们观察到 Spt5 直接与 Pol I 的必需转录起始因子 Rrn3 和核糖体 RNA 结合。基于这些数据，我们提出了一个模型，即 Spt5 在转录早期被招募到 rDNA 中，并提出它通过直接结合 Pol I 复合物在核糖体 RNA 合成中发挥重要作用。
