Differential inhibition of 30S and 70S translation initiation complexes on leaderless mRNA by kasugamycin

In contrast to canonical mRNAs, translation of leaderless mRNA has been previously reported to continue in the presence of the antibiotic kasugamycin. Here, we have studied the effect of the antibiotic on determinants known to affect translation of leadered and leaderless mRNAs. Kasugamycin did not affect the Shine-Dalgarno (SD)-anti-SD (aSD) interaction or the function of translation initiation factor 3 (IF3). Thus, the preferential translation of leaderless mRNA in the presence of kasugamycin can neither be attributed to an expanding pool of 30S subunits with a "blocked" aSD nor to a lack of action of IF3, which has been shown to discriminate against translation initiation at 5'-terminal start codons. Using toeprinting, we observed that on leaderless mRNA 70S in contrast to 30S translation initiation complexes are comparatively resistant to the antibiotic. These results taken together with the known preference of 70S ribosomes for 5'-terminal AUGs lend support to the hypothesis that translation of leaderless mRNAs may as well proceed via an alternative initiation pathway accomplished by intact 70S ribosomes.     

Role of mitochondrial ribosome-dependent translation in germline formation in Drosophila embryos

In Drosophila, mitochondrially encoded ribosomal RNAs (mtrRNAs) form mitochondrial-type ribosomes on the polar granules, distinctive organelles of the germ plasm. Since a reduction in the amount of mtrRNA results in the failure of embryos to produce germline progenitors, or pole cells, it has been proposed that translation by mitochondrial-type ribosomes is required for germline formation. Here, we report that injection of kasugamycin (KA) and chloramphenicol (CH), inhibitors for prokaryotic-type translation, disrupted pole cell formation in early embryos. The number of mitochondrial-type ribosomes on polar granules was significantly decreased by KA treatment, as shown by electron microscopy. In contrast, ribosomes in the mitochondria and mitochondrial activity were unaffected by KA and CH. We further found that injection of KA and CH impairs production of Germ cell-less (Gcl) protein, which is required for pole cell formation. The above observations suggest that mitochondrial-type translation is required for pole cell formation, and Gcl is a probable candidate for the protein produced by this translation system.     

Increased kasugamycin sensitivity in Escherichia coli caused by the presence of an inducible erythromycin resistance (erm) gene of Streptococcus pyogenes

Summary An inducible erythromycin resistance gene (erm) of Streptococcus pyogenes was introduced into Escherichia coli by transformation with a plasmid. The recipient E. coli cells were either kasugamycin sensitive (wildtype) or kasugamycin resistant (ksgA). The MIC values of erythromycin increased from 150 g/ml to>3000 g/ml for E. coli. An extract of transformed cells, particularly a high-salt ribosomal wash, contained an enzyme that was able to methylate 23S rRNA from untransformed cells in vitro; however, 23S rRNA from transformed cells was not a substrate for methylation by such an extract. 165 rRNA and 30S ribosomal subunits of either the wild type or a kasugamycin resistant (ksgA) mutant were not methylated in vitro. Transformation of E. coli by the erm-containing plasmid led to a reduction of the MIC values for kasugamycin. This happened in wild-type as well as in ksgA cells. However, in vitro experiments with purified ksgA encoded methylase demonstrated that also in erm transformed E. coli, the ksgA encoded enzyme was active in wild-type, but not in ksgA cells. It was also shown by in vitro experiments that ribosomes from erm ksgA cells have become sensitive to kasugamycin. Our experiments show that in vivo methylation of 23S rRNA, presumably of the adenosine at position 2058, leads to enhanced resistance to erythromycin and to reduced resistance to kasugamycin. This, together with previous data, argues for a close proximity of the two sites on the ribosome that are substrates for adenosine dimenthylation.Abbreviations MLS macrolide, lincosamide, streptogramin B     

Increased translational fidelity caused by the antibiotic kasugamycin and ribosomal ambiguity in mutants harbouring the ksgA gene

The aminoglycoside kasugamycin, which has previously been shown to inhibit initiation of protein biosynthesis in vitro, also affects translational accuracy in vitro. This is deduced from the observation that the drug decreases the incorporation of histidine relative to alanine into the coat protein of phage MS2, the gene of which is devoid of histidine codons. The read-through of the MS2 coat cistron, due to frameshifts in vitro, is also suppressed by the antibiotic. In contrast, streptomycin enhances histidine incorporation and read-through in this system. The effects of kasugamycin take place at concentrations that do not inhibit coat protein biosynthesis. Kasugamycin-resistant mutants (ksgA) lacking dimethylation of two adjacent adenosines in 16 S ribosomal RNA, show an increased leakiness of nonsense and frameshift mutants (in the absence of antibiotic). They are therefore phenotypically similar to previously described ribosomal ambiguity mutants (ram).