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.

OBJECTIVE: Nonsense mutation readthrough is used as a gene-specific treatment in some genetic diseases. The response to readthrough treatment is determined by the readthrough efficiency of various nonsense mutations. In this manuscript, we aimed to explore the harmful effects of nonsense mutation suppression.
METHODS: HEK293 cells were transfected with two SCN5A (encode cardiac Na+ channel) nonsense mutations, p.R1623X and p.S1812X. We applied two readthrough-enhancing methods (either aminoglycosides or a siRNA-targeting eukaryotic release factor eRF3a (a GTPase that binds eRF1)) to suppress these SCN5A nonsense mutations. When either of readthrough methods was used, the sodium channel proteins were examined by western blot and immunoblotting and recorded by whole cell patch-clamp to observe the functional characterization of the restored channels.
RESULTS: Upon readthrough treatment, the sodium currents were restored to the mutant cDNAs. These mutations reduced full-length sodium channel protein levels, and the sodium currents were reduced to 3% of wild-type. The mutant cDNA sodium currents were increased to 30% of wild-type, and the fulllength proteins also increased. However, the functional characterization of these channels from cDNAs carrying p.R1623X and p.S1812X exhibited abnormal biophysical properties, including a negative shift in steady-state sodium channel inactivation, a positive shift in sodium channel activation and robust late sodium currents. The ramp test showed prolonged QT intervals.
CONCLUSIONS: These results demonstrated that readthrough-enhancing methods effectively suppressed nonsense mutations in SCN5A and restored the expression of full-length channels. However, the restored channels may increase the risk of arrhythmia.