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Related Concept Videos

Ribosome Profiling02:24

Ribosome Profiling

3.7K
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...
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Leaky Scanning02:28

Leaky Scanning

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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...
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Translational Regulation01:29

Translational Regulation

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Translational regulation in prokaryotes ensures efficient protein synthesis by controlling ribosome access to mRNA. This regulation is mediated by secondary RNA structures, including translational riboswitches, RNA thermometers, and small RNAs (sRNAs), which respond to intracellular and environmental signals to modulate gene expression.Translational RiboswitchesRiboswitches in the leader region of mRNAs can regulate translation by altering the accessibility of the Shine-Dalgarno (SD) sequence,...
136
RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Improving Translational Accuracy02:07

Improving Translational Accuracy

11.9K
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...
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Real Time RT-PCR02:57

Real Time RT-PCR

61.4K
Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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Related Experiment Video

Updated: Sep 28, 2025

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling
12:57

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Published on: December 21, 2017

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Normalized Ribo-Seq for Quantifying Absolute Global and Specific Changes in Translation.

Katharina Hoerth1,2, Sonja Reitter1,2, Johanna Schott1,2

  • 1Division of Biochemistry, Mannheim Institute for Innate Immunoscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.

Bio-Protocol
|March 28, 2022
PubMed
Summary
This summary is machine-generated.

Ribosome profiling (Ribo-Seq) can now measure global translation changes. Adding yeast lysate as a spike-in allows absolute quantification of ribosome density, overcoming previous limitations in detecting overall translation suppression or activation.

Keywords:
Normalized ribosome densityRegulation of translationRibo-seqRibosome profilingSpike-in

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RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing
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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
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Last Updated: Sep 28, 2025

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling
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Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

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RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing
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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

Published on: February 18, 2022

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Area of Science:

  • Molecular Biology
  • Genomics
  • Translational Control

Background:

  • Ribosome profiling (Ribo-Seq) quantifies ribosome occupancy on mRNAs genome-wide.
  • Current Ribo-Seq methods determine relative ribosome densities (RD), limiting absolute translation change assessments.
  • Existing protocols cannot distinguish global translation suppression or activation between samples.

Purpose of the Study:

  • To modify the Ribo-Seq protocol for absolute measurement of global translation efficiency.
  • To enable simultaneous detection of relative and global changes in ribosome density.
  • To overcome the limitations of current Ribo-Seq in assessing overall translation regulation.

Main Methods:

  • Modification of the Ingolia et al. (2009) Ribo-Seq protocol.
  • Inclusion of yeast lysate as a spike-in for normalization of sequencing reads.
  • Comparative analysis of ribosome-protected fragments (footprints) and input RNA samples.

Main Results:

  • The modified protocol allows for absolute measurement of global ribosome density (RD) changes.
  • Normalized ribosome density values enable detection of global translation differences between samples.
  • Relative RD changes for specific transcripts can still be determined alongside global shifts.

Conclusions:

  • The addition of a yeast spike-in to Ribo-Seq enables absolute quantification of translation efficiency.
  • This methodological advancement allows for a more comprehensive understanding of translational regulation.
  • The modified Ribo-Seq protocol provides a powerful tool for studying global and gene-specific translation changes.