ИСТИНА

A common toe-print assay implies cDNA synthesis from the primer bound at the 3’-end of the template. It allows determining of the downstream edge of ribosomal or protein complex located on the mRNA. However, such macromolecular complexes as a ribosome cover dozens of nucleotides and their upstream edges are different. The number of covered nucleotides can characterize various functional states of the molecule. Therefore, determining both upstream and downstream coordinates of the complexes is important for studying their activity. The issue has been successfully resolved by using molecule-template crosslinks that stall revertase in the place of contact, and, recently, by ribosome profiling. However, both technics are rather redundant just for the upstream coordinate determination. Here, we present the more simple method based on XRN-1 exonuclease activity. XRN-1 processively cleaves mRNA in the 5’-3’ direction until gets stopped by the ribosome. Then, the ribosome is removed from the template by a temperature treatment or denaturants action, with the simultaneous degradation of XRN-1. The released template is further analyzed by a common primer extension reaction. Using the method in the reconstituted mammalian translation system, we were able to detect the upstream edge of the POST ribosome that, in combination with a standard toe-print assay, gave us the ribosome covered mRNA of 30 nucleotides, that is in good agreement with literature data. The method can be used to investigate detailed ribosome-template dynamics in vitro. The work is supported by RFBR fund №16-34-00406