INDUCIBLE ANTIBIOTIC RESISTANCE

这项研究计划的长期总体目标是研究 对由抗生素诱导或修饰其表达的基因的调节。 重点放在一组赋予抗生素耐药性的基因上。这个 这项建议的具体目的是研究对 红霉素诱导的对大环内酯类、林可胺和 金黄色葡萄球菌产生的链球菌B(MLS)抗生素 表达载体pEl94。一种可诱导的23S rRNA甲基酶通过以下方式产生抗性 将23S rRNA中的特异腺嘌呤残基甲基化，其合成为 红霉素诱导的。进入MLS控制的因素 抗性可以根据它们是否作用于：(1)比率 信息的合成(转录控制)，(2)信息合成的速度 消息的降级(信使核糖核酸的稳定性)，(3)效率 消息的利用(翻译控制)，以及(4)消息的数量 可用于转录消息的质粒DNA副本 (质粒拷贝数控制)。这个问题的两个新方面将 作为当前工作的重点包括：(1)研究了 酶家族成员ErmC甲基酶之间的相互作用 它的23S rRNA底物赋予了MLS抗性；以及(2)对 PEl94Cop蛋白与其可能的结合部位的相互作用 在质粒DNA中，一种被提出用来调节基因表达的反应。 控制质粒拷贝数对红霉素的耐药性。在第一个 案例中，我们将尝试区分 甲基酶氨基酸序列负责的能力 识别23S rRNA底物并将其与其他潜在功能区分开来 底物，如16S rRNA，以及识别 负责催化(即甲基酶)活性的序列。在……里面 第二种情况，我们将研究最近的 鉴定pEl94表达的Cop蛋白及其可能的靶点 质粒pEl94 DNA-明确地问，Cop蛋白结合在哪里 这种绑定最终如何有助于控制 质粒DNA？

The long-term general objective of this research program is to study regulation of genes whose expression is induced or modified by antibiotics. The emphasis is on a group of genes that confer antibiotic resistance. The specific aims of this proposal are to study regulation of erythromycin-inducible resistance to the macrolide, lincosamide, and streptogramin B (MLS) antibiotics conferred by Staphylococcus aureus plasmid pEl94. An inducible 23S rRNA methylase confers resistance by methylating a specific adenine residue in 23S rRNA, and its synthesis is induced by erythromycin. Factors that enter into the control of MLS resistance can be classified according to whether they act on: (1) the rate of synthesis of the message (transcriptional control), (2) the rate of degradation of the message (mRNA stability), (3) the efficiency of utilization of the message (translational control), and (4) the number of plasmid DNA copies that are available from which to transcribe the message (plasmid copy number control). Two new aspects of the problem that will serve as the focus of the present work include: (1) a study of the interaction between the ErmC methylase, a member of the family of enzymes that confer MLS resistance, with its 23S rRNA substrate; and (2) a study of the interaction between the pEl94 Cop protein and its putative binding site in plasmid DNA, a reaction that is proposed to regulate the expression of erythromycin resistance by controlling plasmid copy number. In the first case, we shall attempt to distinguish between structural aspects of the methylase amino acid sequence that are responsible for the ability to recognize the 23S rRNA substrate and distinguish it from other potential substrates such as 16S rRNA as well as to identify the parts of the sequence that are responsible for catalytic (i.e., methylase) activity. In the second case, we shall study the interaction between a recently identified Cop protein specified by plasmid pEl94 and its presumed target plasmid pEl94 DNA - asking specifically, where does Cop protein bind and how does this binding ultimately contribute to control the concentration of plasmid DNA?