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Various effects of paromomycin on tmRNA-directed trans-translation
Toshiharu Takahashi1, Takayuki Konno, Akira Muto
1Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Hirosaki University, Hirosaki 036-8561 Japan.
Abstract:
trans-Translation is an unusual translation in which tmRNA plays a dual function as a tRNA and an mRNA to relieve the stalled translation on the ribosome. In this study, we examined the effects of an aminoglycoside antibiotic, paromomycin, on several tmRNA-related events in vitro. The results of a chemical footprinting study indicated that paromomycin molecules bind tmRNA at G332/G333 in the tRNA domain and A316 in the middle of the long helix between tRNA and mRNA domains. Paromomycin bound at G332/G333 inhibited aminoacylation, and the inhibition was suppressed by the addition of SmpB, a tmRNA-binding protein. It was also found that paromomycin causes a shift of the translation resuming point on tmRNA by -1. The effect on initiation shift was canceled by a mutation at the paromomycin-binding site in 16 S rRNA but not by mutations in tmRNA. A high concentration of paromomycin inhibited trans-translation, whereas it enhanced the initiation-shifted trans-translation when SmpB was exogenously added or a mutation was introduced at 333. The effect of paromomycin on trans-translation differs substantially from that on canonical translation, in which it induces miscoding by modulating the A site of the decoding helix of the small subunit RNA of the ribosome.
Insights
Paromomycin, an antibiotic, binds to tmRNA and affects its function in trans-translation. This binding can inhibit essential processes but is modulated by other factors like SmpB protein.
Area of Science:
- Molecular Biology
- Microbiology
- Biochemistry
Background:
- Trans-translation is a crucial cellular process that rescues stalled ribosomes.
- tmRNA acts as both a transfer RNA (tRNA) and messenger RNA (mRNA) in this process.
- Aminoglycoside antibiotics like paromomycin are known to interfere with bacterial translation.
Purpose of the Study:
- To investigate the in vitro effects of paromomycin on tmRNA-mediated trans-translation.
- To identify specific binding sites of paromomycin on tmRNA.
- To elucidate how paromomycin binding influences key trans-translation events.
Main Methods:
- In vitro biochemical assays.
- Chemical footprinting to map paromomycin binding sites on tmRNA.
- Analysis of aminoacylation and translation resumption.
- Site-directed mutagenesis of tmRNA and 16S rRNA.
Main Results:
- Paromomycin binds tmRNA at G332/G333 and A316, inhibiting aminoacylation.
- SmpB protein can suppress paromomycin-induced inhibition of aminoacylation.
- Paromomycin causes a -1 shift in the translation resumption point on tmRNA.
- This shift is dependent on the paromomycin-binding site in 16S rRNA.
- High paromomycin concentrations inhibit trans-translation, but it can be enhanced under specific conditions (SmpB addition, mutation at G333).
Conclusions:
- Paromomycin directly interacts with tmRNA, affecting its dual role in trans-translation.
- The antibiotic's effects are distinct from its action on canonical translation.
- SmpB and specific tmRNA/rRNA modifications modulate paromomycin's impact on trans-translation.
- These findings provide insights into the complex regulation of bacterial translation and potential antibiotic mechanisms.