Open Access
Biomolecules, volume 10, issue 6, pages 911-19
Ctels: A cell‐free system for the analysis of translation termination rate
Shlyk Valeriya I
1, 2
,
Kushchenko Artem S
1, 3
,
Gladyshev Vadim N.
4
,
Alkalaeva Elena Z
5
,
Dmitriev Sergey E.
1, 3, 5
4
Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
|
Publication type: Journal Article
Publication date: 2020-06-16
PubMed ID:
32560154
Biochemistry
Molecular Biology
Abstract
Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression.
Citations by journals
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1
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3
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Wellcome Open Research
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Wellcome Open Research
3 publications, 23.08%
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1 publication, 7.69%
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Frontiers in Plant Science
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1 publication, 7.69%
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Cells
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Cells
1 publication, 7.69%
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Wiley interdisciplinary reviews. RNA
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Wiley interdisciplinary reviews. RNA
1 publication, 7.69%
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Biochemistry (Moscow)
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1 publication, 7.69%
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Critical Reviews in Biochemistry and Molecular Biology
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Critical Reviews in Biochemistry and Molecular Biology
1 publication, 7.69%
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Nucleic Acids Research
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Nucleic Acids Research
1 publication, 7.69%
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RNA
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RNA
1 publication, 7.69%
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EMBO Reports
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EMBO Reports
1 publication, 7.69%
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3
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Citations by publishers
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1
2
3
4
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Taylor & Francis
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Taylor & Francis
4 publications, 30.77%
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Multidisciplinary Digital Publishing Institute (MDPI)
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Multidisciplinary Digital Publishing Institute (MDPI)
2 publications, 15.38%
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Frontiers Media S.A.
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Frontiers Media S.A.
1 publication, 7.69%
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Wiley
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Wiley
1 publication, 7.69%
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Pleiades Publishing
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Pleiades Publishing
1 publication, 7.69%
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Oxford University Press
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Oxford University Press
1 publication, 7.69%
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Cold Spring Harbor Laboratory
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Cold Spring Harbor Laboratory
1 publication, 7.69%
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Springer Nature
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Springer Nature
1 publication, 7.69%
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1
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4
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- We do not take into account publications that without a DOI.
- Statistics recalculated only for publications connected to researchers, organizations and labs registered on the platform.
- Statistics recalculated weekly.
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Lashkevich K. A. et al. Ctels: A cell‐free system for the analysis of translation termination rate // Biomolecules. 2020. Vol. 10. No. 6. pp. 911-19.
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Lashkevich K. A., Shlyk V. I., Kushchenko A. S., Gladyshev V., Alkalaeva E. Z., Dmitriev S. E. Ctels: A cell‐free system for the analysis of translation termination rate // Biomolecules. 2020. Vol. 10. No. 6. pp. 911-19.
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TY - JOUR
DO - 10.3390/biom10060911
UR - https://doi.org/10.3390%2Fbiom10060911
TI - Ctels: A cell‐free system for the analysis of translation termination rate
T2 - Biomolecules
AU - Shlyk, Valeriya I
AU - Gladyshev, Vadim N.
AU - Alkalaeva, Elena Z
AU - Kushchenko, Artem S
AU - Lashkevich, Kseniya A
AU - Dmitriev, Sergey E.
PY - 2020
DA - 2020/06/16 00:00:00
PB - Multidisciplinary Digital Publishing Institute (MDPI)
SP - 911-19
IS - 6
VL - 10
PMID - 32560154
SN - 2218-273X
ER -
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@article{2020_Lashkevich,
author = {Valeriya I Shlyk and Vadim N. Gladyshev and Elena Z Alkalaeva and Artem S Kushchenko and Kseniya A Lashkevich and Sergey E. Dmitriev},
title = {Ctels: A cell‐free system for the analysis of translation termination rate},
journal = {Biomolecules},
year = {2020},
volume = {10},
publisher = {Multidisciplinary Digital Publishing Institute (MDPI)},
month = {jun},
url = {https://doi.org/10.3390%2Fbiom10060911},
number = {6},
pages = {911--19},
doi = {10.3390/biom10060911}
}
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MLA
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Lashkevich, Kseniya A., et al. “Ctels: A cell‐free system for the analysis of translation termination rate.” Biomolecules, vol. 10, no. 6, Jun. 2020, pp. 911-19. https://doi.org/10.3390%2Fbiom10060911.