Peptidyl-transferase Assay
Peptidyl-transferase Assay - Related Videos
07:46
An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity
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Cited by 3 •
2018
Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.
11:19
Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
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Cited by 2 •
2011
A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.
05:32
Terminal Transferase-Mediated dUTP Nick End-Labeling—TUNEL Assay: An In Situ Method to Detect DNA Fragmentation in Apoptotic Cells
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2025
In this video, the TUNEL assay is performed to detect the presence of apoptotic cells in a sample. The study involves using a special DNA polymerase - terminal deoxynucleotidyl transferase - that catalyzes the addition of labeled dUTPs to the 3' terminus of fragmented DNA, which can be observed and identified under a fluorescence microscope.
14:57
Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases
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Cited by 14 •
2020
Glutathione S-transferases (GSTs) are detoxification enzymes involved in the metabolism of numerous chemotherapeutic drugs. Overexpression of GSTs is correlated with cancer chemotherapy resistance. One way to counter this phenotype is to use inhibitors. This protocol describes a method using a spectrophotometric assay to screen for potential GST inhibitors.
08:12
The TUNEL Assay
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2023
One of the hallmarks of apoptosis is the nuclear DNA fragmentation by nucleases. These enzymes are activated by caspases, the family of proteins that execute the cell death program. TUNEL assay is a method that takes advantage of this feature to detect apoptotic cells. In this assay, an enzyme called terminal deoxynucleotidyl transferase catalyzes the addition of dUTP nucleotides to the free 3’ ends of fragmented DNA. By using dUTPs that are labeled with chemical tags that can produce...
FAQs
The key catalytic event is transfer of the growing peptide from peptidyl-tRNA in the ribosomal P site to aminoacyl-tRNA in the A site. This couples peptide elongation to formation of a longer peptide and leaves the donor tRNA deacylated. Assay results therefore reflect how effectively the ribosome supports this specific step in translation.
Controlled reaction conditions allow individual reaction components or perturbations to be evaluated against peptide-bond formation. Changes in product formation can reveal altered ribosomal activity or catalytic efficiency, while maintaining the same assay framework supports comparisons between samples. This makes the method useful for examining effects associated with ribosomes, tRNAs, or added test compounds.
Antibiotics and ribosome mutations can be examined by comparing product formation under the tested condition with a reference reaction. Reduced or altered activity indicates that the condition affects the measured peptidyl-transferase step, although the assay specifically reports this reaction rather than every event in translation. Such comparisons help investigate ribosome function and compounds targeting bacterial protein synthesis.
A basic setup combines ribosomes with peptidyl-tRNA and aminoacyl-tRNA under controlled conditions, then follows formation of the transferred peptide product. The P-site donor and A-site acceptor provide the substrates for peptide-bond formation. Researchers can vary selected reaction components or test substances while retaining the same product-formation measurement strategy.
Product formation provides a direct experimental readout of the ribosome’s performance in the assayed reaction. Comparing the amount of product generated under different conditions can support estimates of relative catalytic efficiency and reveal whether mutations, antibiotics, or other reaction components change activity. Interpretation should remain tied to the peptidyl-transferase step measured by the assay.
In biology, the assay connects molecular measurements with questions about protein synthesis and ribosome function. It can support studies of how ribosomal mutations alter catalysis, how antibiotics influence bacterial translation, and how candidate compounds affect the reaction targeted during protein synthesis. Measuring product formation under controlled conditions also provides a basis for comparing experimental conditions.