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. (C) 2002 Elsevier Science (USA). All rights reserved.