PREVOTELLA LOESCHEII ADHESIN GENE EXPRESSION

拟议研究的总体目标是澄清 细胞黏附素基因合成的基因表达机制(S) 由口腔细菌Prevoella loescheii产生的产物(SO34蛋白质)。这个 菌毛相关SO34蛋白识别含半乳糖苷 口腔链球菌上的受体34。Plaa基因包含短的和 由150和2310个碱基对组成的长开放阅读框(ORF) 分别进行了分析。粘附素基因的表达需要绕过29 Plaa信使RNA上编码序列的核苷酸(NT)缺口 (M RNA)。编程实现了所提出的Plaa表达机制 移码和核糖体旁路。 符合该机制的间隙区域的特征包括：1) 旁路区域两侧有两个相同的(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) 能够在大的ORF的开始形成一个茎环，以及4) 环中碱基形成假结的能力。建议数 实验将检验Plaa mRNA的上述特征是否 结构对于有效的间隙旁路是必不可少的。站点定向 突变将被用来在可能发挥作用的特定PlaA核苷酸之后 在缝隙旁路中扮演一个角色。一种使用β-半乳糖苷酶报告的化验方法 基因将被用来测量核糖体缺口旁路效率。 此外，这些研究还将为今后的功能研究奠定基础 粘附素作用的研究。高效、稳定的工程表达 SO34粘附素蛋白将促进黏附结合和 可能的是，可以设计的多肽可以防止 粘附素-受体结合。

The overall objective of the proposed research is to elucidate the mechanism(s) of gene expression used to synthesize the plaA adhesin gene product (SO34 protein) by the oral bacterium, Prevotella loescheii. The fimbrial-associated SO34 protein recognizes galactoside-containing receptors on Streptococcus oralis 34. The plaA gene contains short and long open reading frames (ORFs) consisting of 150 and 2310 base pairs (bp) respectively. Expression of the adhesin gene requires bypassing a 29 nucleotide (nt) gap in its coding sequence on the plaA messenger RNA (mRNA). The proposed mechanism of plaA expression is programmed frameshifting and ribosomal bypass. Features of the gap region consistent with this mechanism include: 1) the flanking of the bypass region with two identical (UAA) termination codons; 2) two runs of four or more identical bases (slippery sequences), 3) the ability to form a stem loop at the beginning of the large ORF, and 4) the ability of bases in the loop to form a pseudoknot. The proposed experiments will test whether the above features of the plaA mRNA structure are essential for efficient gap bypass. Site-directed mutagenesis will be used to after specific plaA nucleotides which may play a role in gap bypass. An assay which uses a beta-galactosidase reporter gene will be used to measure ribosomal gap bypass efficiency. In addition, these studies will lay the foundation for future functional studies of adhesin action. Engineering efficient and stable expression of the SO34 adhesin protein will facilitate studies of adhesion binding and possibly, the design of peptides which may be engineered to prevent adhesin-receptor binding.