Narayanan C S, Dubnau D
J Biol Chem. 1987 Feb 5;262(4):1756-65.
We have used a Bacillus subtilis in vitro translation system to test the translational attenuation model for ermC regulation. The ermC gene product is known to methylate rRNA, rendering ribosomes unable to bind this antibiotic. We have shown that the induction of ermC methylase in vitro is post-transcriptional and specific for the macrolides erythromycin and oleandomycin. Erythromycin has no significant effect on the stability of the ermC transcript in vitro, and hence the post-transcriptional induction of methylase under these conditions occurs by stimulation of translation. The induction effect requires ribosomes able to bind erythromycin. By adding small proportions of unmethylated to a methylated extract in the presence of erythromycin, methylase synthesis could be induced. Conversely, when small amounts of methylated extracts were mixed with unmethylated extracts, methylase synthesis could be maintained at elevated levels in the presence of a high concentration of erythromycin. These effects were specific for the inducible ermC, were not observed with a constitutive variant, and could be explained satisfactorily by the translational attenuation model. The roles of three segments of the ermC leader in regulation were explored by probing with appropriate complementary synthetic oligodeoxynucleotides. The induction effect of erythromycin was mimicked by using an oligonucleotide that could free the ribosome binding site for methylase.
我们使用枯草芽孢杆菌体外翻译系统来测试ermC调控的翻译衰减模型。已知ermC基因产物会使核糖体RNA甲基化，使核糖体无法结合这种抗生素。我们已经表明，体外ermC甲基化酶的诱导是转录后水平的，并且对大环内酯类抗生素红霉素和竹桃霉素具有特异性。红霉素在体外对ermC转录本的稳定性没有显著影响，因此在这些条件下甲基化酶的转录后诱导是通过刺激翻译发生的。诱导作用需要能够结合红霉素的核糖体。在红霉素存在的情况下，向甲基化提取物中添加少量未甲基化的提取物，可以诱导甲基化酶的合成。相反，当少量甲基化提取物与未甲基化提取物混合时，在高浓度红霉素存在的情况下，甲基化酶的合成可以维持在较高水平。这些效应对于可诱导的ermC是特异性的，在组成型变体中未观察到，并且可以通过翻译衰减模型得到令人满意的解释。通过用适当的互补合成寡脱氧核苷酸进行探测，研究了ermC前导序列的三个片段在调控中的作用。使用一种能够释放甲基化酶核糖体结合位点的寡核苷酸模拟了红霉素的诱导效应。
