Toeprinting assay explained

The toeprinting assay, also known as the primer extension inhibition assay,^[1] is a method used in molecular biology that allows one to examine the interactions between messenger RNA and ribosomes or RNA-binding proteins.^[2] It is different from the more commonly used DNA footprinting assay. The toeprinting assay has been utilized to examine the formation of the translation initiation complex.^[3]

To do a toeprint assay, one needs the mRNA of interest, ribosomes, a DNA primer, free nucleotides, and reverse transcriptase (RT), among other reagents.^[4] The assay involves letting the RT generate cDNA until it gets blocked by any bound ribosomes, resulting in shorter fragments called toeprints when the results are observed on a sequencing gel.

External links

• (A) Schematic of the primer extension inhibition (toeprint) assay

Notes and References

1. Book: Ribosomes. Methods in Enzymology. 1988-01-01. 0076-6879. 2468068. 419–425. 164. D.. Hartz. D. S.. McPheeters. R.. Traut. L.. Gold. Extension inhibition analysis of translation initiation complexes . 10.1016/s0076-6879(88)64058-4. 978-0-12-182065-7 .
2. Quantitative analysis of ribosome–mRNA complexes at different translation stages. Nucleic Acids Research. 2010-01-01. 0305-1048. 2817456. 19910372. e15. 38. 3. 10.1093/nar/gkp1025. Nikolay E.. Shirokikh. Elena Z.. Alkalaeva. Konstantin S.. Vassilenko. Zhanna A.. Afonina. Olga M.. Alekhina. Lev L.. Kisselev. Alexander S.. Spirin.
3. Inhibition of translation initiation on Escherichia coli gnd mRNA by formation of a long-range secondary structure involving the ribosome binding site and the internal complementary sequence.. Journal of Bacteriology. 1995-11-01. 0021-9193. 177509. 7592434. 6560–6567. 177. 22. J T. Chang. C B. Green. R E. Wolf. 10.1128/jb.177.22.6560-6567.1995.
4. Primer extension analysis of eukaryotic ribosome-mRNA complexes.. Nucleic Acids Research. 1998-11-01. 0305-1048. 147915. 9776744. 4853–4859. 26. 21. M. Kozak. 10.1093/nar/26.21.4853.