Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Improving Translational Accuracy02:07

Improving Translational Accuracy

14.1K
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...
14.1K
Initiation of Translation02:33

Initiation of Translation

38.3K
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...
38.3K
Leaky Scanning02:28

Leaky Scanning

5.6K
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...
5.6K
Termination of Translation01:44

Termination of Translation

27.4K
The large ribosomal subunit has several important structures essential to translation. These include the peptidyl transferase center (PTC) - which is the site where the peptide bond is formed - and a large, internal, water-filled tube through which the nascent polypeptide moves. This latter structure is called the Peptide Exit Tunnel, and it begins at the PTC and spans the body of the large ribosomal subunit. During translation, as the nascent polypeptide chain is synthesized, it passes through...
27.4K
Translation01:31

Translation

17.5K
Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes the final step in the expression of genes. This process is carried out by ribosomes, complexes of protein and specialized RNA molecules. Ribosomes, transfer RNA (tRNA), and other proteins produce a chain of amino acids—the polypeptide—as the end product of translation.
Translation Produces the Building Blocks of Life
Proteins are...
17.5K
Translation01:31

Translation

155.2K
Lesson: Translation
Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes the final step in the expression of genes. This process is carried out by ribosomes, complexes of protein and specialized RNA molecules. Ribosomes, transfer RNA (tRNA), and other proteins produce a chain of amino acids—the polypeptide—as the end product of translation.
Translation Produces the Building Blocks of...
155.2K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

A family of archaeal hibernation factors that bind in tandem and protect ribosomes in dormant cells.

Nature communications·2026
Same author

A family of archaeal hibernation factors that bind in tandem and protect ribosomes in dormant cells.

bioRxiv : the preprint server for biology·2026
Same author

Co-Translational Incorporation of <i>(R)</i>- and <i>(S)</i>-β<sup>2</sup>-Hydroxy Acids <i>In Vitro</i>: A Structural and Biochemical Study on the <i>E. coli</i> Ribosome.

Journal of the American Chemical Society·2026
Same author

Structure and evolution-guided design of minimal RNA-guided nucleases.

bioRxiv : the preprint server for biology·2026
Same author

Translational Downregulation of 5' TOP mRNAs During T Cell Exhaustion.

bioRxiv : the preprint server for biology·2025
Same author

Translon: a single term for translated regions.

Nature methods·2025
Same journal

Layered social competition coordinates reproductive hierarchy formation in ants.

bioRxiv : the preprint server for biology·2026
Same journal

Combination epigenetic-targeted therapy increases the immunogenicity of poorly immunogenic sarcomas.

bioRxiv : the preprint server for biology·2026
Same journal

Loss of LanC-like proteins delays post-injury regeneration of aging skeletal muscles.

bioRxiv : the preprint server for biology·2026
Same journal

Integrative Transfer Network: Deep Transfer Learning Across Populations and Prediction Targets.

bioRxiv : the preprint server for biology·2026
Same journal

Confidence-supported label-free metabolic imaging with FPhaS phase autofluorescence microscopy.

bioRxiv : the preprint server for biology·2026
Same journal

Sequence-encoded autoinhibition couples mRNA decapping activity to phase separation.

bioRxiv : the preprint server for biology·2026
See all related articles

Related Experiment Video

Updated: Jan 13, 2026

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells
08:47

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Published on: May 1, 2020

3.3K

Overcoming the eIF2α Brake in Human Cell-Derived Translation Systems.

Nikolay A Aleksashin1,2, Rohan R Shelke2,3, Tianhao Yin2,4

  • 1Innovative Genomics Institute, University of California, Berkeley, CA, USA.

Biorxiv : the Preprint Server for Biology
|January 7, 2026
PubMed
Summary
This summary is machine-generated.

Inhibitory phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) limits human cell-free translation. Strategies like genome editing or GADD34/K3L expression overcome this block, enhancing synthetic biology applications.

Keywords:
Expi293FGADD34K3LPKRWI-38cardiomyocytecell-free protein synthesiseIF2α phosphorylationiPSCin vitro translationtranslation initiation control

More Related Videos

Xenopus laevis as a Model to Identify Translation Impairment
10:24

Xenopus laevis as a Model to Identify Translation Impairment

Published on: September 27, 2015

11.1K
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

13.0K

Related Experiment Videos

Last Updated: Jan 13, 2026

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells
08:47

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Published on: May 1, 2020

3.3K
Xenopus laevis as a Model to Identify Translation Impairment
10:24

Xenopus laevis as a Model to Identify Translation Impairment

Published on: September 27, 2015

11.1K
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

13.0K

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Synthetic Biology

Background:

  • Cell-free translation systems derived from human cells are crucial for studying gene expression and developing synthetic biology tools.
  • Productivity in these systems is often limited by the inhibitory phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) at the Ser52 residue.

Purpose of the Study:

  • To systematically explore and compare strategies for bypassing the eIF2α phosphorylation-mediated initiation block in both editable and hard-to-edit human cell types.
  • To identify optimal methods for generating high-activity human cell-free translation extracts.

Main Methods:

  • Genome editing of EIF2S1 to create an eIF2α-S52A mutant in Expi293F cells.
  • Genetic knockout of EIF2AK2 (PKR) in Expi293F cells.
  • Stable piggyBac integration of truncated GADD34 (PPP1R15A) and K3L under Tet-inducible control in iPSCs and primary fibroblasts.
  • Differentiation of engineered iPSCs into cardiomyocytes for extract production.

Main Results:

  • Genome editing to block eIF2α Ser52 phosphorylation (eIF2α-S52A) in Expi293F cells significantly increased translation extract activity.
  • Knockout of EIF2AK2 (PKR) also enhanced translation in Expi293F lysates, confirming eIF2α phosphorylation as a key bottleneck.
  • Expression of GADD34 and K3L via piggyBac system in iPSCs (including cardiomyocytes) and primary fibroblasts successfully improved translation output.
  • These expression-based methods provide a viable alternative for systems where genome editing is not feasible.

Conclusions:

  • eIF2α phosphorylation is a primary barrier to robust translation in human cell-free extracts.
  • Genome editing of eIF2α or PKR knockout are optimal for editable cell systems.
  • A portable GADD34/K3L expression cassette enables the production of translationally active lysates from challenging or non-editable systems, broadening the utility of human cell-free translation.