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Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation
Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Taylor & Francis
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8632105/ https://www.ncbi.nlm.nih.gov/pubmed/33938388 http://dx.doi.org/10.1080/15476286.2021.1913312 |
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author | Kim, Changil Holm, Mikael Mandava, Chandra Sekhar Sanyal, Suparna |
author_facet | Kim, Changil Holm, Mikael Mandava, Chandra Sekhar Sanyal, Suparna |
author_sort | Kim, Changil |
collection | PubMed |
description | Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNA(Phe), with the natural dipeptidyl fMet-Phe-tRNA(Phe) in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg(2+) concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA. |
format | Online Article Text |
id | pubmed-8632105 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Taylor & Francis |
record_format | MEDLINE/PubMed |
spelling | pubmed-86321052021-12-01 Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation Kim, Changil Holm, Mikael Mandava, Chandra Sekhar Sanyal, Suparna RNA Biol Research Paper Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNA(Phe), with the natural dipeptidyl fMet-Phe-tRNA(Phe) in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg(2+) concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA. Taylor & Francis 2021-05-03 /pmc/articles/PMC8632105/ /pubmed/33938388 http://dx.doi.org/10.1080/15476286.2021.1913312 Text en © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Paper Kim, Changil Holm, Mikael Mandava, Chandra Sekhar Sanyal, Suparna Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation |
title | Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation |
title_full | Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation |
title_fullStr | Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation |
title_full_unstemmed | Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation |
title_short | Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation |
title_sort | optimization of a fluorescent-mrna based real-time assay for precise kinetic measurements of ribosomal translocation |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8632105/ https://www.ncbi.nlm.nih.gov/pubmed/33938388 http://dx.doi.org/10.1080/15476286.2021.1913312 |
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