CODING GAP BYPASS AND ADHESION FUNCTION IN PREVOTELLA

拟议的研究地址1）使用的独特机制 通过口腔细菌Prevotella loescheii 1295合成 SO 34粘附素蛋白; 2）粘附素的作用机制。 由plaA基因编码的SO 34粘附素是一种凝集素， 像蛋白质，识别含半乳糖苷的受体， 口腔链球菌34细胞。 PlaA表达需要绕过 PlaA上编码序列中的29个核苷酸（nt）缺口 信使RNA（mRNA）。 PlaA的作用机制 表达式是编程的移码跳跃。 特征 与该机制一致的间隙区域包括：1）存在 旁路区侧翼的两个（UAA）终止密码子，2） 四个或更多个相同碱基的两次运行（滑动序列），3） plaA mRNA在起始时形成茎环的能力 的大ORF，和4）在环中的碱基形成的潜力 假结 拟议研究的具体目标是： 使用定点突变和随机突变来测试 plaA mRNA结构的上述特征对于 有效的间隙旁路。 使用β-半乳糖苷酶的测定 报告基因将用于测量编码间隙旁路 效率 B）表达SO 34粘附素并定位其活性位点。 C）在普氏菌中转移和表达plaA粘附素，和/或 用于基因调控和粘附素功能研究的拟杆菌。 这些实验将大大扩展我们对 与牙菌斑相关的口腔细菌的遗传机制。 具体来说，拟议中的实验将阐明不寻常的 用于绕过plaA编码间隙的翻译机制。 plaA基因在相关细菌中的表达将有助于 测试编码空位对plaA表达的影响。 的 拟议的研究将为理解 粘附素结合的机制以及随后的 可以阻止粘附素作用的肽。 了解粘附素 如何防止粘附素与受体结合，将为我们提供一个新的研究方向。 为今后开发预防牙菌斑的药物奠定了基础 积累

The proposed research addresses 1) the unique mechanisms used by the oral bacterium Prevotella loescheii 1295 to synthesize the SO34 adhesin protein, and 2) the mechanisms of adhesin action. The SO34 adhesin, which is encoded by the plaA gene, is a lectin- like protein that recognizes galactoside-containing receptors on Streptococcus oralis 34 cells. PlaA expression requires bypassing a 29-nucleotide (nt) gap in its coding sequence on the PlaA messenger RNA (mRN). The proposed mechanism of PlaA expression is a programmed frameshifting hop. Features of the gap region consistent with this mechanism include: 1) the presence of two (UAA) termination condons flanking the bypass region, 2) two runs of four or more identical bases (slippery sequences), 3) the ability the plaA mRNA to form a stem-loop at the beginning of the large ORF, and 4) the potential of bases in the loop to form a pseudoknot. Specific Aims of the proposed research are to: A0 Use site-directed and random mutagenesis to test whether the above features of the plaA mRNA structure are essential for efficient gap bypass. An assay that uses a beta-galactosidase reporter gence will be used to measure coding gap bypass efficiency. B) Express the SO34 adhesin and map its active site. C) Transfer and express the plaA adhesin in Prevotella and/or Bacteroides for studies of gene regulation and adhesin function. These experiments will significantly expand our understanding of genetic mechanisms in oral bacteria associated with dental plaque. Specifically, the proposed experiments will elucidate the unusual translation mechanisms used to by-pass the plaA coding gap. Expression of the plaA gene in related bacteria will facilitate testing the effect of the coding gap on plaA expression. The proposed studies will provide a solid foundation for understanding mechanisms of adhesin binding and the subsequent engineering of peptides that may prevent adhesin action. Understanding adhesin action and how to prevent adhesin binding to receptors will lay a foundation for the future development of agent that prevent plaque accumulation.