A Ribosomal Density-Mapping Procedure to Explore Ribosome Positions Along Translating mRNAs

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.