Ribosome-bound Rna Purification
Ribosome-bound Rna Purification - Related Videos
02:30
Isolation of Small Regulatory RNA–Bound Bacterial Target RNA Using Affinity Purification
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2025
Source: Mercier, N., et. al., MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria. J. Vis. Exp. (2021)This video demonstrates the isolation of MS2-tagged small regulatory RNA (sRNA) bound to its bacterial target RNA using affinity purification with a maltose-binding protein (MBP)–MS2 coat protein fusion immobilized on amylose resin.
02:53
Ribosomal RNA Synthesis
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2020
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. Ribosome biogenesis begins with the synthesis of 5S and 45S pre-rRNAs by distinct RNA polymerases. The primary transcripts are extensively processed and modified before they are bound and folded by ribosomal proteins and assembly factors,...
09:42
Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides
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Cited by 6 •
2012
We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.
09:13
Chromatographic Purification of Highly Active Yeast Ribosomes
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Cited by 7 •
2011
Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.
06:21
Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq
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Cited by 20 •
2013
A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.
FAQs
Preserving ribosome–RNA complexes during cell lysis helps retain the transcripts that were associated with ribosomes before the sample was fractionated. If those associations are disrupted, the recovered RNA may no longer represent translation-engaged transcripts. This preservation step therefore connects the biochemical preparation to measurements of ribosome occupancy and translational regulation under the cellular condition being studied.
Centrifugation-based separation divides the lysate into fractions enriched for ribosomal or polysomal material, creating a physical basis for selecting RNA associated with translation machinery. The relevant fraction can then be processed separately from other cellular components. Fraction choice directly affects which ribosome-associated transcripts enter the final preparation and therefore influences interpretation of occupancy and translation-related measurements.
Changes in cellular conditions can alter which transcripts remain associated with ribosomes, even when total cellular RNA measurements do not capture the same pattern. Comparing purified ribosome-bound RNA across conditions can therefore reveal shifts in translational regulation and protein synthesis. The resulting data help distinguish changes linked to transcript engagement with ribosomes from broader changes in the cellular RNA pool.
An effective workflow preserves complexes during cell lysis, separates ribosomal or polysomal fractions by centrifugation or a related fractionation method, and releases the RNA from the selected material. The liberated RNA is then extracted for downstream analysis. Maintaining this order helps keep the purification focused on transcripts that were physically associated with ribosomes during preparation.
Purified material can support transcript identification, translation profiling, and examination of ribosome occupancy. These readouts provide complementary views of which RNA molecules are associated with translation machinery and how that association changes. In biochemistry studies, the material can thus connect RNA–ribosome interactions with translational regulation and broader changes in gene expression.
By focusing analysis on RNA associated with ribosomes, the method examines a layer of gene expression linked to protein synthesis rather than only the total RNA pool. This makes it useful for investigating how RNA–ribosome interactions shape translational output. Researchers can apply the approach to compare cellular conditions and identify changes in translation-related organization.