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Fluorescent toeprinting to study the dynamics of ribosomal complexes

Egorova, Tatiana, Sokolova, Elizaveta, Shuvalova, Ekaterina, Matrosova, Vera, Shuvalov, Alexey, Alkalaeva, Elena
Methods 2019 v.162-163 pp. 54-59
complementary DNA, messenger RNA, oligonucleotides, proteins, reverse transcription, ribosomes
Classical toeprinting is generally used to determine the position of ribosomes on mRNA; however, it has several disadvantages. We describe a fluorescent toeprinting assay that enables easier identification of ribosomal complexes bound to mRNA in vitro. The procedure involves the use of stable and safe fluorescently labeled oligonucleotides for reverse transcription reactions as primers, followed by the analysis of cDNA products using an automatic sequencer. This procedure allows the multiplexing and simultaneous analysis of a large number of samples. Over the past ten years, fluorescent toeprinting was applied to determine the activities of eukaryotic release factors and additional proteins involved in translation termination, to study the dynamics of translation initiation and elongation complexes, and to quantitatively evaluate the observed ribosomal complexes. Because of the simplicity and small amounts of material required, fluorescent toeprinting provides a highly scalable and versatile tool to study ribosomal complexes.