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

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.

Blasticidin S inhibits mammalian translation and enhances production of protein encoded by nonsense mRNA

“…Hence, our in vitro experiments indicate that BlaS inhibits elongation more efficiently than peptide release in Mammalia (Figure 1A , C). Consistently, a recent study using an adapted luciferase system to monitor in vitro mammalian translation termination in real-time reported that BlaS (at high concentration) strongly inhibits translation elongation ( 55 ). However, the authors found no effect on termination ( 55 ).…”

“…In eukaryotes, depending on co-translational folding of the nascent peptide the ribosomal exit tunnel covers ∼30–70 amino acid residues of the growing peptide chain ( 54 ). Firefly luciferase C-terminally extended by a respective number of amino acid residues can be enzymatically active while still associated with the ribosome whereas luciferase without C-terminal extension is only active after release from the ribosome ( 55 ). In our reporter system, the Renilla luciferase portion of the Renilla -HBB-WT and NS39 proteins contain C-terminal extensions of 149 and 41 amino acids, respectively ( 29 ).…”

Termi-Luc: a versatile assay to monitor full-protein release from ribosomes

“…1, 2), indicating that varying stop codon identities and downstream sequences can be tested. While our manuscript was under review, firefly luciferase was reported to capture the effects of different 3' UTR sequences and other factors on termination in cell-free translation (Lashkevich et al 2020), highlighting that distinct luciferase reporters can be harnessed for quantitative termination analyses. Furthermore, sequences upstream of the stop codon can be introduced in our method to study the proposed effects of the ORF on termination (Pierson et al 2016).…”

A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis

“…Among those are two universal inhibitors of the peptidyl transferase reaction -bactobolin A (isocoumarin deriva tive) [41,42] and blasticidin S (nucleoside antibiotic) [14,17,43,44]. Interestingly, in bacteria, blasticidin S prima rily inhibits translation termination rather than elonga tion [45], but in eukaryotes, its effect on termination is negligible [46]. A number of insufficiently studied blasti cidin like nucleoside antibiotics, such as anthelmycin (hikizimycin), gougerotin, amicetin, bamicetin, and oth ers [47 49], also weaken aminoacyl tRNA binding and prevent transpeptidation [43].…”

“…However, very few specific termination inhibitors with a well characterized mechanism of action are known (fig ure, t.2). Although reported to specifically block termina tion in bacteria [45], blasticidin S primarily affects the elongation stage in eukaryotes [46]. Another antibiotic, apidaecin (insect antimicrobial peptide), interacts with the bacterial ribosome and arrests translation at the stop codon [144]; however, there is no information on its activity in the eukaryotic systems.…”