Toeprinting Assays: Mapping by Blocks to Reverse Transcriptase Primer Extension

Primer extension inhibition assays, toeprinting, have been used to examine formation of the translational initiation complex (1–16) as well as autogenous regulation of gene expression (17–27) and nucleic acid secondary structure (6, 28–33). A detailed protocol for examining complex formation between ribosome and tRNA at the mRNA ribosome-binding site (RBS) was first published by Hartz et al. (1). Modifications of the toeprint procedure to examine formation of the binary complex between ribosome and RBS were also published by Hartz et al. (6) and Ringquist et al. (7) as have protocols for examining formation of the translational initiation complex in the presence of the translational initiation factors 1, 2, and 3 (3,8,34). Recently, the toeprint method has been used to examine complex formation between RNA and RNA-binding proteins (35–37).