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Related Concept Videos

Initiation of Translation02:33

Initiation of Translation

Initiating translation is complex because it involves multiple molecules. Initiator tRNA, ribosomal subunits, and eukaryotic initiation factors (eIFs) are all required to assemble on the initiation codon of mRNA. This process consists of several steps that are mediated by different eIFs.
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
Initiation of Translation02:33

Initiation of Translation

Initiating translation is complex because it involves multiple molecules. Initiator tRNA, ribosomal subunits, and eukaryotic initiation factors (eIFs) are all required to assemble on the initiation codon of mRNA. This process consists of several steps that are mediated by different eIFs.
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
Leaky Scanning02:28

Leaky Scanning

During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R stands for...
Improving Translational Accuracy02:07

Improving Translational Accuracy

Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
Regulated mRNA Transport02:22

Regulated mRNA Transport

In eukaryotes, transcription and translation are compartmentalized; an mRNA is first synthesized in the nucleus and then selectively transported to the cytoplasm for protein synthesis. Before transport, a pre-mRNA undergoes several steps of post-transcriptional modifications including splicing, 5' capping, and the addition of a poly-adenine tail. Various proteins bind to the pre-mRNA during these modifications. The mRNA transport takes place with the help of multiple proteins playing specific...
Ribosome Profiling02:24

Ribosome Profiling

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...

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Related Experiment Video

Updated: Jul 6, 2026

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs
10:37

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Published on: May 10, 2018

Cap-dependent eukaryotic initiation factor-mRNA interactions probed by cross-linking.

Lisa Lindqvist1, Hiroaki Imataka, Jerry Pelletier

  • 1Department of Biochemistry, Faculty of Medicine, McGill University, Montreal, Quebec H3G 1Y6, Canada.

RNA (New York, N.Y.)
|March 28, 2008
PubMed
Summary
This summary is machine-generated.

Researchers investigated how proteins bind to the 5' cap of messenger RNA (mRNA) during translation initiation. Using a novel method, they identified interactions of eukaryotic initiation factor 4G (eIF4G), eIF4H, and eIF3 with mRNA near the cap structure.

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An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics
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An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

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Related Experiment Videos

Last Updated: Jul 6, 2026

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs
10:37

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Published on: May 10, 2018

mRNA Interactome Capture from Plant Protoplasts
12:29

mRNA Interactome Capture from Plant Protoplasts

Published on: July 28, 2017

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics
09:52

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Published on: September 15, 2020

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Cap-dependent translation initiation is crucial for eukaryotic gene expression.
  • The eukaryotic initiation factor (eIF) 4F complex, including eIF4E, eIF4A, and eIF4G, plays a key role in recruiting ribosomes to mRNA.
  • Previous studies used chemical and UV cross-linking to understand initial protein-mRNA interactions.

Purpose of the Study:

  • To develop and apply a novel splint-ligation-mediated approach for studying protein-mRNA interactions during cap-dependent translation.
  • To characterize the cross-linking of proteins adjacent to and downstream from the 7-methyl guanosine cap structure.
  • To identify specific eukaryotic initiation factors involved in the cap recognition process.

Main Methods:

  • A splint-ligation-mediated approach was employed to generate mRNA containing 4-thiouridine.
  • A radiolabel group was incorporated adjacent to the 4-thiouridine for cross-linking studies.
  • Cap-dependent cross-linking of proteins to mRNA was monitored using the generated mRNA construct.

Main Results:

  • The study successfully generated 4-thiouridine-containing mRNA for cross-linking analysis.
  • The approach allowed monitoring of protein cross-linking adjacent to and downstream from the mRNA cap structure.
  • Interactions between eIF4G, eIF4H, and eIF3 subunits with mRNA during cap recognition were demonstrated.

Conclusions:

  • The developed splint-ligation method provides a valuable tool for studying protein-mRNA interactions in translation initiation.
  • New insights into the roles of eIF4G, eIF4H, and eIF3 in cap-dependent ribosome recruitment were obtained.
  • This research refines our understanding of the molecular mechanisms governing the initiation of eukaryotic translation.