Toeprinting assay

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The toeprinting assay (originally called extension inhibition assay(1)) is method to look at the interaction of messenger RNA with ribosomes or other RNA-binding proteins. It is different from standard DNA footprinting assay. The toeprinting assay identifies only one edge of the complex on the RNA, whereas DNA footprinting identified both edges of the complex on the DNA. The toeprinting assay has been utilized to examine the formation of the translation initiation complex. The general idea is that you take an RNA of interest and mix it with the 30s subunit of the ribosome, initiator tRNA, a synthetic DNA primer that hybridizes downstream of the binding site, the four deoxynucleotide triphosphates, and reverse transcriptase (RT). RT will normally synthesize cDNA by extending the primer to the 5' end of the RNA. However, a bound ribosome will block RT from continuing, resulting in a shortened cDNA that is called a toeprint when observed on a sequencing gel. The precise nucleotide position of the toeprint on the RNA can be determined by running a DNA sequencing reaction generated with the same primer in adjacent lanes.

1. Hartz, D., McPheeters, D.S., Traut, R. and Gold, L. 1988. Extension inhibition analysis of translation initiation complexes. Methods in Enzymology 164, 419-425.