Molecular mechanismn of transcription termination factor Rho as a hexameric RAN/DNA helicase

急诊大肠杆菌转录终止蛋白Rho是一种六聚体解旋酶，据信其功能是以ATP依赖性方式分离RNA-DNA杂合体。为了进一步阐明Rho的分子机制，我们针对Rho与F1-ATP酶的结构相似性，进行了以下两方面的研究：1）我们先前的交联研究表明Rho六聚体具有伪C3对称性，其亚基以交替的方式呈现双共形状态。我们在此进一步尝试通过用羟胺消化来绘制Rho上的交联位置，所述羟胺单独切割Asn 151和Gly 152之间的rho多肽。结果表明，在所有亚基-亚基界面处，来自一个亚基的N-末端片段上的赖氨酸与来自相邻亚基的C-末端片段上的赖氨酸交联。在另一条带上，Rho六聚体的三维重建预测Lysl 23位于邻近亚基上的Lys 224和Lys 249附近。因此，Lys 123将成为与Lys 224或Lys 2492交联的良好候选者。为了确定Rho的确切三维结构，我们还进行了X射线晶体学分析。最初，我们只能获得质量差的Rho晶体，没有显示出清晰的衍射。通过反复试验，我们确定了几种可以改善Rho晶体质量的条件：a）加入高浓度的KCl（0 5 M）; B）延长晶体老化时间; c）赖氨酸残基的甲基化;等等.然而，即使将这些条件结合起来，迄今为止所制得的最好晶体的衍射分辨率也不令人满意，最多为10埃.最近，我们发现添加配体如ATP类似物和oligo（dC）有助于将Rho蛋白保持在单分散状态，这是制备良好晶体的先决条件。因此，正在进行试验以使含有这些配体的络合物结晶

The E. coli transcription termination protein Rho is a hexameric helicase, and is believed to function by separating an RNA-DNA hybrid in an ATPdependent manner. To further elucidate the molecular mechanism of Rho, we have focused on its structural similarity to F1-ATP-ase and conducted the following two lines of studies1) Our previous cross-linking study suggested that the Rho hexamer has a pseudo-C3 symmetry, in which its subunits take dual conformanonal states in an alternating manner. We further tried herein to map the positions of cross-links on Rho by digestion with hydroxylamine, which singly cuts the rho polypeptide between Asn 151 and Gly 152. The result revealed that a lysine on the N-terminal fragment from one subunit is cross-linked to a lysine on the C-terminal fragment from an adjacent subunit at all subunit-subunit interfaces. On the other band, a three-dimensional reconstruction of Rho hexamer predicted that Lysl23 is positioned in close proximity to Lys224 and Lys249 on an adjacent subunit. Thus, Lysl23 would make a good candidate for crosslinks to Lys224 or Lys2492) Toward determining the exact three-dimensional structure of Rho, we also set out for its X-ray crystallographic analysis. Initially, we could only obtain Rho crystals of poor qualities that showed no clear diffractions. Through subsequent repeated trials, we have identified several conditions that could improve the quality of Rho crystal: a) addition of a high concentration of KC1 (0 5 M); b) a prolonged aging of crystal; c) methylation of lysine residues; and so on. Even with these conditions combined, however, best crystals so far made yielded diffractions at unsatisfactory resolutions of at most 10 Angstrom. Most recently, we have found that the addition of ligands such as ATP analogs and oligo (dC) help to maintain Rho protein in a mono-disperse state, a prerequisite for making good crystals. Thus trials are in progress to crystallize complexes containing such ligands