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

大肠杆菌转录终止蛋白Rho是一种六聚解旋酶，被认为以atp依赖的方式分离RNA-DNA杂交体。为了进一步阐明Rho的分子机制，我们重点研究了其与f1 - atp酶的结构相似性，并进行了以下两方面的研究：1)我们之前的交联研究表明Rho六聚体具有伪c3对称，其亚基以交替的方式呈现双共形态。我们进一步尝试用羟胺消化来绘制Rho上交联的位置，羟胺在Asn 151和Gly 152之间切割Rho多肽。结果表明，一个亚基的n端片段上的赖氨酸在所有亚基-亚基界面上都与相邻亚基的c端片段上的赖氨酸交联。另一方面，Rho六聚体的三维重建预测Lysl23位于邻近亚基上的Lys224和Lys249附近。因此，Lysl23将成为Lys224或Lys2492交联的良好候选者)为了确定Rho的确切三维结构，我们还着手对其进行x射线晶体学分析。最初，我们只能得到质量差的Rho晶体，没有明显的衍射。通过随后的反复试验，我们确定了几个可以提高Rho晶体质量的条件：a)加入高浓度的KC1 (0.5 M)；B)晶体老化时间延长；C)赖氨酸残基甲基化；等等......。然而，即使把这些条件结合起来，迄今为止制造的最好的晶体也只能产生令人不满意的衍射，其分辨率至多为10埃。最近，我们发现配体如ATP类似物和寡聚物（dC）的添加有助于维持Rho蛋白处于单分散状态，这是制造良好晶体的先决条件。因此，将含有这种配体的配合物结晶化的试验正在进行中

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