A BACTERIAL GROUP II INTRON INVOLVED IN CONJUGATION

本申请寻求对第一组II内含子L1.1trB的分子和遗传分析的支持，该内含子在原核生物体内显示出功能。II组内含子通常存在于真核生物的细胞器中，并通过类似于真核细胞核基因中剪接体内含子的机制进行剪接。它们也是移动的元件，可以通过新的反转录转座机制在基因组中易位。因此，这些元件的分子生物学与真核生物和病毒的基因调控和进化的理解有关。在PI实验室与合作研究者合作研究乳酸乳球菌质粒pRSO 1的接合质粒转移过程中发现了L1.1trB。该内含子的剪接是编码接合松弛酶的外显子基因ltrB的功能性基因产物表达所必需的。在所提出的实验中，剪接依赖的缀合系统将被利用在内含子剪接的分子和遗传分析的组合。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。在这一目标的一个主要重点将是参与相互作用的内含子序列与内含子编码的蛋白质（IEP）和成熟酶功能的遗传和分子基础上赋予的IEP。- 分析L1.1trB剪接功能的调节，并检查剪接和缀合功能协调调节的程度。- 使用剪接依赖性结合分析鉴定和分析小分子剪接抑制剂。

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.