Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
J Biochem. 2022 Jul 25;172(2):79-88. doi: 10.1093/jb/mvac043.
Komagataella pastoris is a methylotrophic yeast that is commonly used as a host cell for protein production. In the present study, we reconstituted the nucleosome with K. pastoris histones and determined the structure of the nucleosome core particle by cryogenic electron microscopy. In the K. pastoris nucleosome, the histones form an octamer and the DNA is left-handedly wrapped around it. Micrococcal nuclease assays revealed that the DNA ends of the K. pastoris nucleosome are somewhat more accessible, as compared with those of the human nucleosome. In vitro transcription assays demonstrated that the K. pastoris nucleosome is transcribed by the K. pastoris RNA polymerase II (RNAPII) more efficiently than the human nucleosome, while the RNAPII pausing positions of the K. pastoris nucleosome are the same as those of the human nucleosome. These results suggested that the DNA end flexibility may enhance the transcription efficiency in the nucleosome but minimally affect the nucleosomal pausing positions of RNAPII.
毕赤酵母是一种甲醇营养型酵母，通常被用作蛋白质生产的宿主细胞。在本研究中，我们用毕赤酵母组蛋白重新组装核小体，并通过低温电子显微镜确定了核小体核心颗粒的结构。在毕赤酵母核小体中，组蛋白形成八聚体，DNA 呈左手螺旋缠绕在其上。微球菌核酸酶实验表明，与人类核小体相比，毕赤酵母核小体的 DNA 末端更易接近。体外转录实验表明，毕赤酵母核小体由毕赤酵母 RNA 聚合酶 II（RNAPII）转录的效率高于人类核小体，而毕赤酵母核小体的 RNAPII 暂停位置与人类核小体相同。这些结果表明，DNA 末端的灵活性可能会提高核小体中的转录效率，但对 RNAPII 的核小体暂停位置的影响很小。
