CODING GAP BYPASS AND ADHESION FUNCTION IN PREVOTELLA

这项拟议的研究涉及1)使用的独特机制 由口腔细菌Prevoella loescheii1295合成 SO34粘附素蛋白；2)粘附素的作用机制。 SO34粘附素是一种凝集素，由PlaA基因编码。 类似于识别含半乳糖苷受体的蛋白质 口腔链球菌34细胞。Plaa表达式需要绕过 其编码序列在平原上有29个核苷酸(NT)的缺口 信使核糖核酸(MRN)。拟议中的Plaa机制 表情是一种程序化的移帧跳跃。的功能 符合该机制的间隙区域包括：1)存在 旁路区域两侧的两个(UAA)终止密码子，2) 四个或更多相同碱基的两个序列(滑动序列)，3) Plaa信使核糖核酸在一开始形成茎环的能力 以及4)环中碱基形成的潜力 一个假结。拟议研究的具体目标是：A0 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 高效的缝隙旁路。一种使用β-半乳糖苷酶的分析方法 将使用报告收敛来测量编码间隙旁路 效率。B)表达SO34粘附素并绘制其活性部位图。 C)在Prevoella和/或中转移和表达Plaa粘附素 类杆菌用于基因调控和粘附素功能的研究。 这些实验将极大地扩展我们对 口腔细菌与牙菌斑相关的遗传机制。 具体地说，拟议中的实验将阐明不寻常的 用于绕过Plaa编码间隙的转换机制。 PlaA基因在相关细菌中的表达将有助于 测试编码间隙对Plaa表达的影响。这个 拟议的研究将为理解 粘附素结合机制及后续工程研究进展 可能阻止粘附素作用的多肽。了解粘连 作用以及如何防止粘附素与受体结合将为 为未来斑块防腐剂的发展奠定基础 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.