Various effects of paromomycin on tmRNA-directed trans-translation.
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.
Takahashi
Toshiharu
T
Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Hirosaki University, Hirosaki 036-8561 Japan.
Konno
Takayuki
T
Muto
Akira
A
Himeno
Hyouta
H
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010303:Paromomycin / Q000494:pharmacology*; D011072:Poly U; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D000900:Anti-Bacterial Agents; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; C096222:tmRNA; D011072:Poly U; D010303:Paromomycin