Crucifix Corinne, Uhring Muriel, Schultz Patrick
Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP 1, rue Laurent Fries, BP10142 67404 Illkirch, France.
J Struct Biol. 2004 Jun;146(3):441-51. doi: 10.1016/j.jsb.2004.02.001.
The structural study of transient nucleoprotein complexes by electron microscopy is hampered by the coexistence of multiple interaction states leading to an heterogeneous image population. To tackle this problem, we have investigated the controlled immobilization of double stranded DNA molecules and of nucleoprotein complexes onto a support suitable for cryo-electron microscopy observation. The DNA was end-labeled with a biotin moiety in order to decorate, or to be incorporated into, two-dimensional streptavidin crystals formed in contact of a biotinylated lipid layer. The binding specificity and efficiency were examined by radioactively labeled oligonucleotides and by direct visualization of unstained and hydrated nucleic acid molecules in cryo-electron microscopy. By using RNA polymerase we further show that, once immobilized, femtomolar amounts of DNA template are suitable to interact with the enzyme. The image analysis of the RNA polymerase-DNA complexes showed that a three-dimensional model can be retrieved from such samples.
通过电子显微镜对瞬时核蛋白复合物进行结构研究，因多种相互作用状态共存导致图像群体异质性而受到阻碍。为解决这一问题，我们研究了将双链DNA分子和核蛋白复合物可控固定在适合冷冻电子显微镜观察的支持物上的方法。DNA用生物素部分进行末端标记，以便修饰或掺入在生物素化脂质层接触处形成的二维链霉亲和素晶体中。通过放射性标记的寡核苷酸以及在冷冻电子显微镜下直接观察未染色和水合的核酸分子，检测了结合特异性和效率。通过使用RNA聚合酶，我们进一步表明，一旦固定，飞摩尔量的DNA模板就适合与该酶相互作用。RNA聚合酶-DNA复合物的图像分析表明，可以从此类样品中获取三维模型。
