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.
Highlights
TmRNA is widely distributed among eubacteria and has been found in some chloroplasts and possibly in some mitochondria [1,2,3,4]
We first found that paromomycin, a neomycin-class aminoglycoside antibiotic, significantly increased the melting temperature of E. coli tmRNA (Table 1)
The band of DMS modification at A316 and the bands of KE modification at G332 and G333 disappeared with an increase of the paromomycin concentration (Fig. 1b)
Summary
TmRNA (transfer-messenger RNA, known as 10Sa RNA or SsrA RNA) is widely distributed among eubacteria and has been found in some chloroplasts and possibly in some mitochondria [1,2,3,4]. It is a novel molecule with both tRNA and mRNA properties. It was found on other polypeptides when they are translated from artificial mRNAs lacking a termination codon [4] or possessing a cluster of rare codons [13] and from endogenous mRNAs in E. coli and Bacillus subtilis [14,15,16]
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