Localization of in vivo ribosome pause sites

A protocol for the localization of the 5′ boundaries of in vivo ribosomal pausing sites has been developed. These mapping experiments combine two basic techniques. The first is the isolation of polysomal transcripts via centrifugation of tissue extracts through a sucrose cushion in the presence of translational elongation inhibitors. The second technique involves a micrococcal nuclease protection assay first developed by Wolin and Walter for in vitro-bound ribosomes (EMBO J. 7, 3559-3569, 1988). Using this method, the 5′ boundaries of in vivo ribosomal pause sites were localized on spinach chloroplast mRNAs derived from the atpA gene. This method is easily adaptable to the identification of in vivo ribosomal pause sites from any organism. It could also be adapted to the localization of in vivo binding sites for other nucleic acid binding proteins.