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

Autor: Valeriya I Shlyk, Vadim N. Gladyshev, Elena Alkalaeva, Kseniya A Lashkevich, Sergey E. Dmitriev, Artem S Kushchenko
Rok vydání: 2020
Předmět:
eRF1
eRF3
Termination factor
DNA Mutational Analysis
lcsh:QR1-502
nascent peptide release
In Vitro Techniques
eRF1(AGQ) mutant
Biochemistry
Article
lcsh:Microbiology
3′ UTR length
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
Eukaryotic translation
Mutation Rate
Genes
Reporter
Protein biosynthesis
Humans
blasticidin S
Luciferase
Eukaryotic release factors
Luciferases
Molecular Biology
030304 developmental biology
0303 health sciences
stop codon read-through
Cell-Free System
translation termination
Translation (biology)
eukaryotic release factors
Peptide Chain Termination
Translational
firefly luciferase
in vitro translation system
Stop codon
Blasticidin S
Cell biology
chemistry
Codon
Nonsense
Protein Biosynthesis
Codon
Terminator
Biological Assay
030217 neurology & neurosurgery
Peptide Termination Factors
Zdroj: Biomolecules, Vol 10, Iss 911, p 911 (2020)
Biomolecules
Volume 10
Issue 6
ISSN: 2218-273X
DOI: 10.3390/biom10060911
Popis: 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&prime
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 &ldquo
leaky&rdquo
stop codon context, which likely defines the basis of nonsense suppression.
Databáze: OpenAIRE
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