Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB Canada.
RNA Biol. 2012 Oct;9(10):1288-301. doi: 10.4161/rna.22131. Epub 2012 Sep 20.
YchF is one of two universally conserved GTPases with unknown cellular function. As a first step toward elucidating YchF's cellular role, we performed a detailed biochemical characterization of the protein from Escherichia coli. Our data from fluorescence titrations not only confirmed the surprising finding that YchFE.coli binds adenine nucleotides more efficiently than guanine nucleotides, but also provides the first evidence suggesting that YchF assumes two distinct conformational states (ATP- and ADP-bound) consistent with the functional cycle of a typical GTPase. Based on an in vivo pull-down experiment using a His-tagged variant of YchF from E. coli (YchFE.coli), we were able to isolate a megadalton complex containing the 70S ribosome. Based on this finding, we report the successful reconstitution of a YchF•70S complex in vitro, revealing an affinity (KD) of the YchFE.coli•ADPNP complex for 70S ribosomes of 3 μM. The in vitro reconstitution data also suggests that the identity of the nucleotide-bound state of YchF (ADP or ATP) modulates its affinity for 70S ribosomes. A detailed Michaelis-Menten analysis of YchF's catalytic activity in the presence and the absence of the 70S ribosome and its subunits revealed for the first time that the 70S ribosome is able to stimulate YchF's ATPase activity (~10-fold), confirming the ribosome as part of the functional cycle of YchF. Our findings taken together with previously reported data for the human homolog of YchF (hOLA1) indicate a high level of evolutionary conservation in the enzymatic properties of YchF and suggest that the ribosome is the main functional partner of YchF not only in bacteria.
YchF 是两种普遍保守的 GTPase 之一，其细胞功能未知。为了阐明 YchF 的细胞作用，我们首先从大肠杆菌中对该蛋白进行了详细的生化特性分析。我们的荧光滴定数据不仅证实了一个令人惊讶的发现，即 YchF.E.coli 比鸟嘌呤核苷酸更有效地结合腺嘌呤核苷酸，而且还首次提供了证据表明 YchF 假定两种不同的构象状态（ATP 和 ADP 结合）与典型 GTPase 的功能循环一致。基于使用来自大肠杆菌的 His 标记变体（YchF.E.coli）进行的体内下拉实验，我们能够分离出含有 70S 核糖体的兆道尔顿复合物。基于这一发现，我们报告了成功在体外重建 YchF•70S 复合物，揭示了 YchF.E.coli•ADPNP 复合物与 70S 核糖体的亲和力（KD）为 3 μM。体外重建数据还表明，YchF 的核苷酸结合状态（ADP 或 ATP）的身份调节其与 70S 核糖体的亲和力。在存在和不存在 70S 核糖体及其亚基的情况下，对 YchF 催化活性的详细米氏分析首次表明，70S 核糖体能够刺激 YchF 的 ATP 酶活性（~10 倍），证实核糖体是 YchF 功能循环的一部分。我们的发现与之前报道的 YchF 的人同源物（hOLA1）的数据相结合，表明 YchF 的酶学特性具有高度的进化保守性，并表明核糖体不仅是细菌中 YchF 的主要功能伙伴。
