Eldad Naama, Arava Yoav
Department of Biology, Technion - Israel Institute of Technology, Haifa, Israel.
Methods Mol Biol. 2008;419:231-42. doi: 10.1007/978-1-59745-033-1_16.
The number and distribution of ribosomes on a transcript provide useful information in ascertaining the efficiency of translation. Herein we describe a direct method to determine the association of ribosomes with specific regions of an mRNA. The method, termed Ribosome Density Mapping (RDM), includes cleavage of ribosomes-associated mRNAs with RNase H and complementary oligodeoxynucleotide followed by separation of the cleavage products on a sucrose gradient. The gradient is then fractionated and the sedimentation position of each mRNA fragment is determined by northern analysis. Although developed for yeast mRNAs, RDM is likely to be applicable to various other systems.
转录本上核糖体的数量和分布为确定翻译效率提供了有用信息。在此，我们描述一种直接方法来确定核糖体与mRNA特定区域的关联。该方法称为核糖体密度图谱分析（RDM），包括用核糖核酸酶H和互补寡聚脱氧核苷酸切割与核糖体相关的mRNA，然后在蔗糖梯度上分离切割产物。接着对梯度进行分级分离，并通过Northern分析确定每个mRNA片段的沉降位置。尽管RDM是针对酵母mRNA开发的，但它可能适用于各种其他系统。
