Translational accuracy of a tethered ribosome

Abstract

Ribosomes are evolutionary conserved ribonucleoprotein complexes that function as two separate subunits in all kingdoms. During translation initiation, the two subunits assemble to form the mature ribosome, which is responsible for translating the messenger RNA. When the ribosome reaches a stop codon, release factors promote translation termination and peptide release, and recycling factors then dissociate the two subunits, ready for use in a new round of translation. A tethered ribosome, called Ribo-T, in which the two subunits are covalently linked to form a single entity, was recently described in Escherichia coli 1 . A hybrid ribosomal RNA (rRNA) consisting of both the small and large subunit rRNA sequences was engineered. The ribosome with inseparable subunits generated in this way was shown to be functional and to sustain cell growth. Here, we investigated the translational properties of Ribo-T. We analyzed its behavior in -1 or +1 frameshifting, stop codon readthrough, and internal translation initiation.Our data indicate that covalent attachment of the two subunits modifies the properties of the ribosome, altering its ability to initiate and terminate translation correctly.

Aminoglycoside tolerance inVibrio choleraeengages translational reprogramming associated with queuosine tRNA modification

“…Growth in the presence of tobramycin at 50% of the MIC (0.2 µg/ml in MH); NT: non-treated. Reporters described in (33). Y axis represents stop codon readthrough percentage*10.…”

“…To test whether a defect in Q modification influences the fidelity of translation in the presence and absence of sub-MIC TOB, previously developed reporter tools were used (33), to measure stop codons readthrough and ribosomal frameshifting in V. cholerae ∆tgt and wild-type strains. The system consists of vectors containing readthrough or frameshift-promoting signals inserted between the lacZ and luc sequences, encoding β-galactosidase and luciferase, respectively.…”

“…V. cholerae electrocompetent cells were transformed with reporter dual reporter plasmids that were previously described (33). Overnight cultures of transformants were diluted 1:100 in MH medium supplemented with 5 μg/mL chloramphenicol to maintain plasmids, 200 μg/mL IPTG (Isopropyl β-D-1thiogalactopyranoside) and in the presence or not of tobramycine 0,4 μg/mL and grown with shaking at 37°C until an OD600 of 0.3 (exponential phase).…”

“…TOB: sub-MIC tobramycin treatment (0.2 µg/ml). Reporters described in (33). Y axis represents frameshifting percentage.…”