Cell-Free Applications: RNA Toeprinting

A protein chain being produced from a ribosome.
Precise mapping of the positions of ribosomes and associated factors on mRNAs is essential for characterizing the mechanism of translation. Using the toeprinting assay, mRNA is translated using purified components or crude cell lysates such as rabbit reticulocyte. Cycloheximide is added to the reaction to inhibit elongation. This arrests the position of the ribosomes on the mRNA transcript. The mRNA/ribosomal complex are then copied into cDNA by reverse transcriptase using a complementary radiolabeled primer. Where the reverse transcriptase meets the ribosome bound to the mRNA, cDNA extension is halted, and a toeprint cDNA fragment is generated.

The following references use rabbit reticulocyte lysates as the basis for toeprinting experiments to better understand the mechanism of translation.

Weill, L. et al. (2010)Nucl. Acid, Res. 38, 1367–81. A combination of chemical/enzymatic analyses indicated that gag open reading frame of three viruses adopts a stable secondary structure that allows IRES mediated translation. Mutations that destabilized conserved elements severely inhibit translation. Additional analysis via toeprinting showed HIV-2 IRES has the unique ability to attract up to three initiation complexes on a single RNA molecule.

De Breyne, S. et al. (2008) RNA 14, 367–80. The Simian picornavirus type 9 (SPV9) genome contains a group of IRES that resembles hepacivirus/pestvirus (HP) IRES. Characterization of the initiation process using the toeprinting assay in correlation with other techniques revealed aspects that resemble initiation on the HP IRES and others that are unique to SPV9.

Andreev, D. et al. (2008) RNA 14, 233–39. Rel E is a well characterized toxin involved in the nutritional stress response in bacteria and archae. Rel lacks any eukaryote homolog. Based on toeprinting data, it was demonstrated that RelE cleaves mRNA in the A site of the eukaryote ribosome.

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Gary Kobs

Gary Kobs

Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and Sr. Product Manager for Protein Analysis products. Gary has retired from Promega Corporation.

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