PREVOTELLA LOESCHEII ADHESIN GENE EXPRESSION

拟议研究的总体目标是阐明 用于合成plaA粘附素基因的基因表达机制 产品（SO 34蛋白）的口腔细菌，普氏菌loescheii。的 菌毛相关SO 34蛋白识别含半乳糖苷的 口腔链球菌34. plaA基因含有短的， 由150和2310个碱基对（bp）组成的长开放阅读框（ORF） 分别粘附素基因的表达需要绕过A29 plaA信使RNA上编码序列中的核苷酸（nt）缺口 （mRNA）。plaA表达的机制被编程 移码和核糖体旁路。 与该机制一致的差距区域的特征包括：1） 旁路区侧翼有两个相同的（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） 在大ORF的开始处形成茎环的能力，以及4） 环中碱基形成假结的能力。拟议 实验将测试plaA mRNA的上述特征是否 结构对于有效间隙旁路是必要的。定点 诱变将用于在特定的plaA核苷酸后， 在间隙旁路中的作用。使用β-半乳糖苷酶报告基因的测定 基因将用于测量核糖体间隙旁路效率。 此外，这些研究将为今后的功能性 粘附素作用的研究。工程化高效稳定表达 SO 34粘附素蛋白将促进粘附结合研究， 可能的是，肽的设计可以被工程化以防止 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.