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首页> 外文期刊>Applied and Environmental Microbiology >Is the In Situ Accessibility of the 16S rRNA of Escherichia coli for Cy3-Labeled Oligonucleotide Probes Predicted by a Three-Dimensional Structure Model of the 30S Ribosomal Subunit?
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Is the In Situ Accessibility of the 16S rRNA of Escherichia coli for Cy3-Labeled Oligonucleotide Probes Predicted by a Three-Dimensional Structure Model of the 30S Ribosomal Subunit?

机译：是否通过30S核糖体亚基的三维结构模型预测Cy16标记的寡核苷酸探针对大肠杆菌16S rRNA的原位可及性？

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Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-?-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.
机译：荧光标记的，rRNA靶向的寡核苷酸杂交的系统研究表明，原位可及性存在很大差异。对靶位点可及性的可靠预测将有助于更合理地设计探针，以鉴定自然环境中的单个微生物细胞。在过去的三年中，对核糖体高阶结构的大量研究使我们对核糖体的空间构象有了进一步的了解。这些研究的范围从基于协变分析的rRNA-rRNA相互作用的鉴定到核糖体的物理成像以鉴定蛋白质-rRNA的相互作用。在这里，我们就核糖体小亚基的三级结构模型，重新评估了我们的大肠杆菌16S rRNA原位可达性数据。我们在30S核糖体亚基的3.0-α分辨率三维（3D）模型上定位了176个寡核苷酸的靶序列。探针杂交效率与探针靶区域与30S核糖体亚基模型表面的接近程度之间几乎没有相关性。我们将其归因于以下事实：在含有变性核糖体的固定细胞上进行了荧光原位杂交，而核糖体的3D模型基于其天然构象。测试了不同的固定和杂交方案对一组10个代表性探针产生的荧光信号的影响。与从低聚甲醛到乙醇的固定剂变化相比，存在或不存在强变性洗涤剂十二烷基硫酸钠的影响要明显得多。

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