Local Tertiary Structure Probing of Ribonucleoprotein Particles by Nuclease Fusion Proteins

Analyses of the conformational dynamics of the numerous cellular ribonucleoprotein particles (RNP) significantly contribute to the understanding of their modes of action. Here, we tested whether ribonuclease fusion proteins incorporated into RNPs can be used as molecular probes to characterize the local RNA environment of these proteins. Fusion proteins of micrococcal nuclease (MNase) with ribosomal proteins were expressed in S. cerevisae to produce in vivo recombinant ribosomes which have a ribonuclease tethered to specific sites. Activation of the MNase activity by addition of calcium led to specific rRNA cleavage events in proximity to the ribosomal binding sites of the fusion proteins. The dimensions of the RNP environment which could be probed by this approach varied with the size of the linker sequence between MNase and the fused protein. Advantages and disadvantages of the use of MNase fusion proteins for local tertiary structure probing of RNPs as well as alternative applications for this type of approach in RNP research are discussed.

对多种细胞核糖核蛋白颗粒（ribonucleoprotein particles, RNP）的构象动态进行分析，对阐明其作用模式具有重要意义。本研究旨在探究整合至核糖核蛋白颗粒中的核糖核酸酶融合蛋白，能否作为分子探针以表征此类蛋白的局部RNA微环境。本研究将微球菌核酸酶（micrococcal nuclease, MNase）与核糖体蛋白的融合蛋白在酿酒酵母（S. cerevisae）中表达，获得了将核糖核酸酶锚定至特定位点的体内重组核糖体。通过添加钙离子激活MNase活性后，可在融合蛋白的核糖体结合位点附近引发特异性的核糖体RNA（ribosomal RNA, rRNA）切割事件。该方法可探测的核糖核蛋白颗粒环境的尺度，随微球菌核酸酶与融合蛋白之间的接头序列长度而变化。本文还讨论了利用微球菌核酸酶融合蛋白进行核糖核蛋白颗粒局部三级结构探测的优缺点，以及该方法在核糖核蛋白颗粒研究中的其他潜在应用方向。