Toeprinting

Toeprinting is a technique that was developed in the translation field to measure precisely the position of ribosomes (or ribosomal subunits) on mRNAs. It relies on the fact that reverse transcriptase is “stopped” when it encounters a blocking complex (ribonucleoprotein or protein) on the RNA. The length of the primer extension product produced when the RNA is occupied by such a complex compared to the full-length primer extension product indicates the 3′ position of the complex of interest. Although primarily used in measuring ribosome occupancy and position, toeprinting can be used in a variety of other contexts. The procedure described here was developed to measure the position (and presence) of the exon junction complex deposited on mRNAs as a consequence of splicing. Toeprinting can, in principle, be used whenever the complex of interest is bound stably enough to withstand displacement of transcribing reverse transcriptase.