Regulation of a Plasmid Gene in Bacillus Subtilis

氯霉素（Cm）通过阻断70S核糖体翻译过程中的肽延伸抑制细菌生长。Cm还在革兰氏阳性菌中作为cat基因（编码氯霉素乙酰转移酶）表达的诱导剂。一个基因cat-86的诱导已被证明是由于mRNA翻译的激活。这似乎是由于Cm能够阻止核糖体翻译cat-86调节先导肽；一个停滞的核糖体被认为破坏了一个下游的RNA茎环，该茎环隔离了cat-86核糖体的结合位点。本提案的长期目标是解释Cm激活cat-86和其他诱导猫基因的机制。预计这些研究将澄清和扩展我们对核糖体靶向抗生素作用机制的理解。这些研究也有望揭示衰减调节模型在控制基因表达方面的原理。通过使用位点定向诱变和重组DNA技术，现在可以确定所提出的Cm失速位点是mRNA还是蛋白质序列，以及该位点的序列。将确定失速位点与受调控RNA二级结构之间的空间关系。细菌中明显对cm不敏感的翻译退出的罕见例子。这些是由于碰巧没有Cm摊位造成的吗？土壤细菌芽孢杆菌属的某些物种对抗生素氯霉素具有天然抗性，因为它们能够合成一种使药物失活的酶。灭活酶的合成是由药物特异性诱导的，也就是说，细菌只有在遇到药物时才会产生这种酶。洛维特博士早期的研究表明，药物引起的核糖体（合成蛋白质的细胞器）行为的改变，是诱导失活酶机制的关键部分。这是调控基因表达的一种非常不寻常的机制，洛维特博士在这项研究中提出要弄清这种机制的许多尚不清楚的细节。除了对一种新的调控机制的见解之外，这项工作还应该对如何控制核糖体的功能产生新的见解。

Chloramphenicol (Cm) inhibits bacterial growth by blocking peptide elongation during translation on 70S ribosomes. Cm also acts as an inducer of the expression of cat genes (encoding chloramphenicol acetyltransferase) in Gram-positive bacteria. Induction of one gene, cat-86, has been shown to be due to activation of mRNA translation. This appears to result from the ability of Cm to stall ribosomes translating a cat-86 regulatory leader peptide; a stalled ribosome is thought to disrupt a downstream RNA stem-loop that sequesters the cat-86 ribosome binding site. The long term goal of this proposal is to explain the mechanism which allows Cm to activate cat-86, and other inducible cat genes. It is anticipated that these studies will clarify and extend our understanding of the mechanism of action of a ribosomally targeted antibiotic. It is also expected that these studies will show the breath of the principles of the attenuation regulatory model in controlling gene expression. By use of site directed mutagenesis and recombinant DNA technology it is now possible to determine whether the proposed Cm stall site is an mRNA or protein sequence, and the sequence of the site. The spatial relationship between the stall site and the regulated RNA secondary structure will be identified. Uncommon examples of apparently Cm-insensitive translation exit in bacteria. Do these result from the fortuitous absence of a Cm stall site? Certain species of the soil bacterium genus Bacillus are naturally resistant to the antibiotic chloramphenicol by virtue of their ability to synthesize an enzyme which inactivates the drug. The synthesis of the inactivating enzyme is specifically inducible by the drug, i.e., the bacteria make the enzyme only when they encounter the drug. Dr. Lovett's earlier studies have shown that a drug-induced change in the behavior of ribosomes, the organelles which synthesize proteins, is a critical part of the mechanism of the induction of the inactivating enzyme. This is a very unusual mechanism for the regulation of gene expression, and Dr. Lovett proposes in this study to work out many of the still obscure details of this mechanism. Apart from insight into a novel regulatory mechanism, this work should also yield new insight into how the function of ribosomes is controlled.