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JoVE Journal

Biochemistry

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Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

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10:37 min

May 10th, 2018

May 10th, 2018

12,323 Views

0:04

Title

0:34

Preparation of mRNA

2:59

Toeprinting Assay

6:32

Sequencing Reactions and Gel Electrophoresis

7:53

Results: Importance of IRES Secondary Structure in the Formation of Translation Initiation Complexes on an Uncapped XIAP IRES RNA

9:35

Conclusion

Transcript

The overall goal of this toeprinting analysis is to observe the translation initiation complex formation on mammalian mRNAs. This method can help answer key questions in the field of mRNA translation, such as IRES mediated translation initiation.

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Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

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