INTEGRATION SPECIFICITY OF THE TY3 RETROTRANSPOSON

Ty3是酿酒酵母中的一种逆转录病毒模型元件，它插入聚合酶III转录的基因的转录起始位点。现在有许多生物化学和遗传工具可用于研究Ty3。这些包括针对Ty3蛋白的抗体，核酸中间体的检测，体内和体外转位检测，以及一大批Ty3转位缺陷的突变体。在拟议的工作中，生命周期中不太为人所知的方面将成为重点，包括整合位点选择和染色体背景对染色体背景下Ty3靶向和表达的影响，易位到细胞核，以及从病毒样颗粒到预整合复合物的转变。具体目标有五个：1)鉴定Ty3位置特异性整合的蛋白质和DNA介质。Ty3的最小体外整合靶标是与靶基因结合的B′。重组试剂可用，包括IN， RT和NC，用于解决整合前复合物的性质。进行直接接触的蛋白质将被识别，蛋白质和DNA结构对靶活性的相对贡献将被确定。2)了解Ty3与染色体之间的相互作用。Ty3被两侧的pol iii转录基因抑制，并且Ty3似乎不能进入位于rDNA重复序列中的染色体5S基因，尽管它很容易使用质粒携带的基因。我们将探讨染色质结构在这些现象中的作用。Ty3作为tRNA基因的绝缘体元件的假设将得到验证。3)鉴定Ty3和Ty3核转运途径的宿主成分。在目前的资助期内，Ty3 NLS定位于IN的羧基末端区域，并且鉴定了可能影响Ty3核易位的宿主突变体。实验将探讨Ty3核定位预整合复合体的组成，该核蛋白复合体是否使用共识单分子基本结构域nls蛋白使用的α - β输入蛋白途径，以及亚核定位是否在Ty3靶向中发挥作用。4)描述了VLP向预积分复杂过渡的信号和介质。逆转录的完成伴随着特定的修饰或重排，这些修饰或重排标志着剥离导致核进入的假设将被验证。5)利用遗传和生化方法鉴定Ty3生命周期中新的宿主功能。宿主突变影响Ty3转位的机制将在一组50个宿主突变中进行研究。将分析vlp预整合复合物的非Ty3蛋白，这些非Ty3蛋白与Ty3复合物存在物理关联，因此可能影响Ty3的生命周期，并将讨论这些蛋白在Ty3生命周期中的作用。

Ty3 is a retrovirus model element in S. cerevisiae that inserts into the transcription initiation site of genes transcribed by polymerase III. Many biochemical and genetic tools are now available with which to study Ty3. These include antibodies against Ty3 proteins, assays for nucleic acid intermediates, in vivo and in vitro assays for transposition, and a large group of mutants defective in Ty3 transposition. In the proposed work, aspects of the lifecycle which are less well understood, will be the focus, including integration site selection and effects of chromosomal context on targeting and expression of Ty3 in the chromosomal context, translocation into the nucleus, and the transition from viruslike particle to preintegration complex. There are five Specific Aims: 1) Identification of protein and DNA mediators of Ty3 position-specific integration. The minimal in vitro integration target for Ty3 is B' bound to the target gene. Recombinant reagents are available, including IN, RT, and NC with which to address the nature of the preintegration complex. The proteins that make direct contacts will be identified and the relative contributions of protein and DNA structures to target activity determined. 2) Understanding interactions between Ty3 and the chromosome. Ty3 is repressed by flanking pol III-transcribed genes and Ty3 appears not to access chromosomal 5S genes located in the rDNA repeat, although it readily uses plasmid-borne genes. The role of chromatin structure in these phenomena will be explored. The hypothesis that Ty3 acts as an insulator element for tRNA genes will be tested. 3) Identification of Ty3 and host components of the Ty3 nuclear translocation pathway. The Ty3 NLS was localized to the carboxyl-terminal region of IN in the current grant period and host mutants were identified which may affect Ty3 nuclear translocation. Experiments will address the composition of the Ty3 nuclear localized preintegration complex, whether this nucleoprotein complex uses the alpha-beta importin pathway used by consensus monopartite basic domain NLS-containing proteins, and whether subnuclear localization plays a role in Ty3 targeting. 4) Delineation of signals and mediators of the VLP to preintegration complex transition. The hypothesis that completion of reverse transcription is accompanied by specific modifications or rearrangements that signal uncoating resulting in nuclear entry will be tested. 5) identification of new host functions in the Ty3 lifecycle using genetic and biochemical approaches. The mechanism by which host mutations affect Ty3 tranposition will be examined for a set of fifty host mutants. The VLP-preintegration complex fraction will be analyzed for non- Ty3 proteins which are physically associated with Ty3 complexes and which may therefore affect the Ty3 lifecycle and the role of those proteins in the Ty3 lifecycle will he addressed.