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Related Concept Videos

Ribosome Profiling02:24

Ribosome Profiling

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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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Improving Translational Accuracy02:07

Improving Translational Accuracy

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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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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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Termination of Translation01:44

Termination of Translation

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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...
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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,...
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Ribosomal RNA Synthesis02:53

Ribosomal RNA Synthesis

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Ribosome synthesis is a highly complex and coordinated process involving more than 200 assembly factors. The synthesis and processing of ribosomal components occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells.
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Related Experiment Video

Updated: Jun 10, 2025

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
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Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

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Versatile Dual Reporter to Identify Ribosome Pausing Motifs Alleviated by Translation Elongation Factor P.

Urte Tomasiunaite1, Tess Brewer1, Korinna Burdack1

  • 1Faculty of Biology, Microbiology, Ludwig-Maximilians-Universität München, 82152 Planegg-Martinsried, Germany.

ACS Synthetic Biology
|October 19, 2024
PubMed
Summary
This summary is machine-generated.

Researchers developed a novel plasmid-based dual reporter system to rapidly screen for amino acid motifs that cause translational pausing in bacteria. This tool identifies new motifs affecting protein synthesis efficiency, aiding in understanding ribosome stalling and translation speed regulation.

Keywords:
dual reporterelongation factor Pproteins with polyproline motifsribosome stallingscreening for ribosome pausing strengthstalling motifstranslational efficiency

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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
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Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
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RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing
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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Protein synthesis speed is regulated by amino acid sequences, with polyproline motifs known to cause ribosome stalling.
  • Translation elongation factor P (EF-P) is crucial for overcoming ribosome stalling at these motifs.
  • Existing methods for identifying pausing motifs are limited in scope and throughput.

Purpose of the Study:

  • To develop and validate a novel plasmid-based dual reporter system for rapid, high-throughput screening of translational pausing motifs.
  • To identify previously unknown amino acid motifs that influence translational efficiency and ribosome dynamics in Escherichia coli.
  • To provide a versatile platform for studying the impact of sequence variations on protein synthesis.

Main Methods:

  • Construction of a dual reporter plasmid encoding mScarlet-I and chloramphenicol acetyltransferase for fluorescence and survival-based screening.
  • Creation and screening of diprolyl and other proline-containing motif libraries in an E. coli strain lacking EF-P.
  • Utilizing fluorescence-associated cell sorting (FACS) for high-throughput screening of motif libraries.

Main Results:

  • The dual reporter system successfully identified motifs with varying pausing strengths in an EF-P deficient E. coli strain.
  • High-throughput screening revealed new amino acid motifs that significantly influence translational efficiency.
  • The system demonstrated efficacy in identifying sequence-dependent effects on protein synthesis.

Conclusions:

  • The developed plasmid-based dual reporter is an effective tool for rapid in vivo screening of translational pausing motifs.
  • This platform facilitates the discovery of novel motifs impacting protein synthesis and ribosome behavior.
  • The findings contribute to a deeper understanding of the regulation of translation speed and efficiency.