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Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs
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Biochemistry
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JoVE Journal Biochemistry
Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

DOI:
10.3791/57519-v

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10:37 min

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May 10, 2018

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1Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute,University of Lethbridge, 2Department of Neuroscience and the Canadian Centre for Behavioral Neuroscience (CCBN),University of Lethbridge, 3Arnie Charbonneau Cancer Institute,University of Calgary

Chapters

  • 00:04Title
  • 00:34Preparation of mRNA
  • 02:59Toeprinting Assay
  • 06:32Sequencing Reactions and Gel Electrophoresis
  • 07:53Results: Importance of IRES Secondary Structure in the Formation of Translation Initiation Complexes on an Uncapped XIAP IRES RNA
  • 09:35Conclusion

Summary

Automatic Translation

Automatic Translation

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.

Tags

Toeprinting AnalysisTranslation Initiation ComplexMammalian MRNAsIRESRNA PurificationRabbit Reticulocyte LysateRNase InhibitorATPGMP-PNPDTTSpermidine

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