A BACTERIAL GROUP II INTRON INVOLVED IN CONJUGATION

这项申请寻求对第一组II内含子L1.1trB的分子和遗传分析的支持，该内含子被证明在原核生物中具有体内功能。第二类内含子经常存在于真核生物的细胞器中，其剪接机制与真核核基因中的剪接体内含子相似。它们也是可移动的元件，可以通过新的逆转座子机制在基因组中移位。因此，这些元件的分子生物学对于理解真核生物和病毒中的基因调控和进化是相关的。L1.1trB是在与研究人员合作研究乳酸乳球菌质粒pRSO1接合转移的过程中在PI实验室发现的。该内含子的剪接是表达外显子基因ltrB的功能基因产物所必需的，该外显子编码一种共轭松弛酶。在拟议的实验中，剪接依赖的共轭系统将被用于内含子剪接的联合分子和遗传分析。具体目标包括：-使用简单的遗传分析来分离和表征影响L1.1trB剪接的突变。这一目标的一个主要焦点将是与内含子编码蛋白(IEP)相互作用的内含子序列，以及由内含子编码蛋白赋予的成熟酶功能的遗传和分子基础。-分析L1.1trB剪接功能的调节，并检查剪接和结合功能协调调节的程度。-使用剪接依赖结合试验鉴定和分析小分子剪接抑制物。

This application seeks support for molecular and genetic analyses of the first group II intron, L1.1trB, shown to be functional in vivo in prokaryotes. Group II introns are often found in organelles of eukaryotes and splice by a mechanism similar to that of spliceosomal introns in eukaryotic nuclear genes. They are also mobile elements which may translocate in genomes by novel retrotransposition mechanisms. Thus the molecular biology of these elements is relevant to the understanding of gene regulation and evolution in eucaryotes and viruses. L1.1trB was discovered in the laboratory of the PI in the course of collaborative research with the coinvestigator on conjugative plasmid transfer of the Lactococcus lactis plasmid pRSO1. Splicing of this intron is required for expression of a functional gene product from the exon gene, ltrB, encoding a conjugative relaxase enzyme. In the proposed experiments, the splicing-dependent conjugation system will be exploited in a combined molecular and genetic analysis of intron splicing. The specific aims include: - Use of simple genetic assays to isolate and characterize mutations affecting L1.1trB splicing. A major focus in this objective will be the intron sequences involved in interaction with the intron-encoded protein (IEP) and on the genetic and molecular basis of the maturase function conferred by the IEPs. - Analysis of the regulation of L1.1trB splicing functions, and examination of the extent to which splicing and conjugation functions are coordinately regulated. - Identification and analysis of small molecule splicing inhibitors using a splicing-dependent conjugation assay.