Ribosome footprinting (aka ribosome profiling aka ribo-seq) is great for seeing what mRNAs ribosomes are bound to in cells. But if you want to see where exactly ribosomes are on a particular mRNA, you need to dip your toes in! 

Ribosome toe printing (aka primer extension inhibition) works by giving ribosomes in a cell-free translation system a template and using reverse transcription with a labeled primer that targets the 3’ end of that template. Let the reverse transcriptase do its thing and it will make a complementary DNA (cDNA) copy of the end of the template until it runs into the ribosome. Then purify those labeled cDNAs and run them alongside sequencing lanes that show you where A, C, T, & G are in the template (you can prepare these by reverse-transcribing the template and spiking in labeled dideoxynucleotide (dead end nucleotides) – 1 letter per reaction – this will cause there to be a range of cDNAs ending in the labeled letter, and you can compare them to your sample. You can do things like add ribosome inhibitors like cycloheximide (CHX) (at concentrations where they prevent elongation but not initiation) to get the ribosomes to build up at start sites so you can see them. And even without adding anything you can see if the ribosome is stalled places already. 

Sometimes ribosomes stall because they’re translating awkward sequences like things with a bunch of prolines. And “programmed” stalling can actually be used by cells as a regulatory mechanism. Sometimes stalling is caused or relieved in the presence of various drugs or metabolites (small molecules that are part of metabolic pathways – so breakdown or build-up products). This stalling often occurs in upstream open reading frames (uORFs) and regulates expression of the main ORF (which has the protein making instructions). A cool example of this is some bacteria making an antibiotic resistance gene, ermC in response to the presence of the corresponding antibiotic, erythromycin. More on that here:

Vazquez-Laslop N, Thum C, Mankin AS. Molecular mechanism of drug-dependent ribosome stalling. Mol Cell. 2008 Apr 25;30(2):190-202. https://doi.org/10.1016/j.molcel.2008.02.026 

And here are some other examples of toeprinting at work…

Here’s the original paper: Hartz…Gold (1988) Extension inhibition analysis of translation initiation complexes, Methods in enzymology http://www.ncbi.nlm.nih.gov/pubmed/2468068

Here’s a paper that uses it in a PURE expression system (where the translation’s happening in a reconstituted system where the minimum required components have been purified and combined) as opposed to a lysate-based system (where you break cells open (lyse them) and use the machinery that was in there, unpurified – and you add some extra factors to make things more efficient). This paper has nice a methods section.

Cédric Orelle, Teresa Szal, Dorota Klepacki, Karen J. Shaw, Nora Vázquez-Laslop, Alexander S. Mankin, Identifying the targets of aminoacyl-tRNA synthetase inhibitors by primer extension inhibition, Nucleic Acids Research, Volume 41, Issue 14, 1 August 2013, Page e144, https://doi.org/10.1093/nar/gkt526  

more on PURE systems here: blog: https://bit.ly/cellfreeexpression

This paper uses it to study stalling at termination codons: Karousis, E.D., Gurzeler, LA., Annibaldis, G. et al. Human NMD ensues independently of stable ribosome stalling. Nat Commun 11, 4134 (2020). https://doi.org/10.1038/s41467-020-17974-z

Here’s a paper describing a fluorescence-based system, where they do sequencing with a capillary gel electrophoresis sequencing machine instead of reading out radiolabeled bands on a slab gel. 

Egorova, T., Sokolova, E., Shuvalova, E., Matrosova, V., Shuvalov, A., & Alkalaeva, E. (2019). Fluorescent toeprinting to study the dynamics of ribosomal complexes. Methods162-163, 54–59. https://doi.org/10.1016/j.ymeth.2019.06.010 

Here’s a paper about a different technique, inverse toeprinting, which goes at things from the 5’ end with sequencing so you can vary the template randomly to see what sequences might cause a stall, and then see what caused that stall. 

High-throughput inverse toeprinting. Britta Seip, Guénaël Sacheau, Denis Dupuy, C Axel Innis. Life Science Alliance Oct 2018, 1 (5) e201800148; DOI: 10.26508/lsa.201800148 https://www.life-science-alliance.org/content/1/5/e201800148 

More on ribosome profiling (aka ribosome footprinting aka RiboSeq) here: https://bit.ly/ribosomefootprinting

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