Centre de Biochimie Structurale, CNRS UMR5048, INSERM U1054, Université de Montpellier I & II, 34090 Montpellier, France.
EMBO Rep. 2013 May;14(5):473-9. doi: 10.1038/embor.2013.39. Epub 2013 Apr 5.
SpoIIIE/FtsK are membrane-anchored, ATP-fuelled, directional motors responsible for chromosomal segregation in bacteria. Directionality in these motors is governed by interactions between specialized sequence-recognition modules (SpoIIIE-γ/FtsK-γ) and highly skewed chromosomal sequences (SRS/KOPS). Using a new combination of ensemble and single-molecule methods, we dissect the series of steps required for SRS localization and motor activation. First, we demonstrate that SpoIIIE/DNA association kinetics are sequence independent, with binding specificity being uniquely determined by dissociation. Next, we show by single-molecule and modelling methods that hexameric SpoIIIE binds DNA non-specifically and finds SRS by an ATP-independent target search mechanism, with ensuing oligomerization and binding of SpoIIIE-γ to SRS triggering motor stimulation. Finally, we propose a new model that provides an entirely new interpretation of previous observations for the origin of SRS/KOPS-directed translocation by SpoIIIE/FtsK.
SpoIIIE/FtsK 是一种膜结合的、由 ATP 驱动的定向马达,负责细菌中的染色体分离。这些马达的方向性由专门的序列识别模块(SpoIIIE-γ/FtsK-γ)和高度倾斜的染色体序列(SRS/KOPS)之间的相互作用控制。使用组合的集合和单分子方法,我们剖析了 SRS 定位和马达激活所需的一系列步骤。首先,我们证明了 SpoIIIE/DNA 结合动力学与序列无关,结合特异性仅由解离决定。接下来,我们通过单分子和建模方法表明,六聚体的 SpoIIIE 非特异性地结合 DNA,并通过一种 ATP 独立的靶标搜索机制找到 SRS,随后寡聚化和 SpoIIIE-γ 与 SRS 的结合触发马达刺激。最后,我们提出了一个新的模型,为 SpoIIIE/FtsK 起源的 SRS/KOPS 定向易位的先前观察结果提供了全新的解释。