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Open AccessArticle

CTELS: A Cell-Free System for the Analysis of Translation Termination Rate

1
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119234 Moscow, Russia
2
Department of Molecular Biology, Biological Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia
3
School of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119234 Moscow, Russia
4
Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
5
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
*
Author to whom correspondence should be addressed.
The first two authors contribute equally to this study.
Biomolecules 2020, 10(6), 911; https://doi.org/10.3390/biom10060911
Received: 11 April 2020 / Revised: 29 May 2020 / Accepted: 13 June 2020 / Published: 16 June 2020
(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)
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. View Full-Text
Keywords: translation termination; nascent peptide release; firefly luciferase; in vitro translation system; eukaryotic release factors; eRF1; eRF3; eRF1(AGQ) mutant; blasticidin S; 3′ UTR length; stop codon read-through translation termination; nascent peptide release; firefly luciferase; in vitro translation system; eukaryotic release factors; eRF1; eRF3; eRF1(AGQ) mutant; blasticidin S; 3′ UTR length; stop codon read-through
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Lashkevich, K.A.; Shlyk, V.I.; Kushchenko, A.S.; Gladyshev, V.N.; Alkalaeva, E.Z.; Dmitriev, S.E. CTELS: A Cell-Free System for the Analysis of Translation Termination Rate. Biomolecules 2020, 10, 911.

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