INTEGRATION SPECIFICITY OF THE TY3 RETROTRANSPOSON

Ty3是S.在一些实施方案中，所述载体是插入到由聚合酶III转录的基因的转录起始位点的酿酒酵母的载体。 现在有许多生物化学和遗传学工具可用于研究Ty3。 这些包括针对Ty3蛋白的抗体、核酸中间体的测定、转座的体内和体外测定以及Ty3转座缺陷的一大组突变体。 在拟议的工作中，生命周期的方面，这是不太好理解，将是重点，包括整合位点的选择和染色体背景的靶向和表达的Ty3在染色体的背景下，易位到细胞核中，从病毒样颗粒的过渡到preintegration复合物的影响。具体目的有五个：1）鉴定Ty3位置特异性整合的蛋白质和DNA介导物。 Ty3的最小体外整合靶标是与靶基因结合的B '。 可获得重组试剂，包括IN、RT和NC，用于解决整合前复合物的性质。 将鉴定进行直接接触的蛋白质，并确定蛋白质和DNA结构对靶活性的相对贡献。 2)了解Ty3和染色体之间的相互作用。 Ty3被抑制的侧翼聚合酶III转录的基因和Ty3似乎不访问位于rDNA重复的染色体5S基因，虽然它很容易使用质粒携带的基因。 染色质结构在这些现象中的作用将被探讨。 将检验Ty3作为tRNA基因的绝缘子元件的假设。 3)Ty3和Ty3核易位途径的宿主组分的鉴定。 Ty3 NLS定位于IN的羧基末端区域，并鉴定了可能影响Ty3核转位的宿主突变体。 实验将解决Ty3核定位的整合前复合物的组成，该核蛋白复合物是否使用由含有NLS的蛋白质的共有单组分碱性结构域所使用的α-β输入蛋白途径，以及亚核定位是否在Ty3靶向中起作用。 4)描绘VLP向整合前复合物过渡的信号和介质。 逆转录的完成伴随着特定的修饰或重排，信号脱壳导致核进入的假设将被测试。 5)使用遗传和生物化学方法鉴定Ty3生命周期中的新宿主功能。将对一组50个宿主突变体检查宿主突变影响Ty3转座的机制。将分析VLP-整合前复合物级分中与Ty3复合物物理相关并因此可能影响Ty3生命周期的非Ty3蛋白，并将阐述这些蛋白在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.