Phage T4 Head Assembly and Initiation of Infection

描述（由申请人提供）：该拨款申请建议继续研究两个长期目标：了解1)保守病毒DNA包装马达的易位机制及其调控；2)为对抗新型DNA易位限制性内切酶而注射的内部蛋白的进化。包装马达由T4大末端酶亚基组成，在前体的入口十二聚体上工作，以接近100%的效率转运任何序列的染料标记短dna。以下是单个复合物，我们将分析门户- gfp前端包装。通过FCS和核酸酶检测，短缺口DNA没有被包装，与气泡错配DNA不同，这表明马达将扭转引入DNA以使其易位。FRET将用于建立与双染料DNA， DNA的尺寸被停止的马达改变。此外，在标记的端酶和入口之间和内部的FRET将在包装过程中建立距离和构象变化。FRET也将用于建立在封装头两端的接近度。我们将确定末端酶的头部切割特异性。我们将完成我们的工作，证明晚期西格玛因子(gp55)-滑动钳（gp45）复合体通过大末端酶亚基gp17与许多调节蛋白相互作用参与了缺口环状dna的体内包装起始。在第二个目标中，通过IPI位点的基因扩增，一个不同的cts注射蛋白家族已经进化出来，以挑战一个新的NTP驱动的DNA易位限制酶家族。我们正在进行的两种gmrS/gmrD限制性内切酶（CT和UT），两种多态噬菌体基因拮抗剂（IPI*和IP5*，其结构我们已经确定），以及这些酶的多种靶糖HMC加合物的分析揭示了在纳米尺度上产生复杂性的进化途径。总的来说，致力于第一个DNA包装目标的努力将比第二个限制性内切酶注射抑制剂部分更重要，这是我们之前资助的两个研究目标。然而，研究DNA易位的方法在两个研究领域将是互补的。

DESCRIPTION (provided by applicant): The grant application proposes continuation of research toward two long term objectives: understanding 1) the translocation mechanism of the conserved viral DNA packaging motor and its regulation; and 2) the evolution of the internal proteins injected to counter novel DNA translocating restriction enzymes. The packaging motor consists of the T4 large terminase subunit working at the portal dodecamer of the prohead to translocate dye labeled short DNAs of any sequence with near 100% efficiency. Following single complexes we will analyze portal-GFP prohead packaging. By FCS and nuclease assay short nicked DNAs are not packaged, unlike bubble mismatch DNAs, suggesting that the motor introduces torsion into the DNA to translocate it. FRET will be used to establish with double dye DNAs that the dimensions of the DNA are changed by the stalled motor. Additionally FRET between and within tagged terminase and portal will establish distance and conformational changes during packaging. FRET will also be used to establish the proximity of the two ends in packaged heads. We will determine the headful cutting specificity of the terminase. We will complete our work showing the participation of the late sigma factor (gp55)-sliding clamp (gp45) complex in packaging initiation in vivo on nicked circular DNAs by large terminase subunit gp17 interaction with a number of regulatory proteins. In the second objective, a diverse family of CTS-injected proteins has evolved by gene expansion at the IPI locus to challenge a novel family of NTP driven DNA translocating restriction enzymes. Our ongoing analysis of two of the gmrS/gmrD restriction endonucleases (CT and UT), two polymorphic phage gene antagonists (IPI* and IP5* whose structures we have determined), and diverse target sugar HMC adducts of these enzymes reveals an evolutionary pathway generating complexity on a nanoscale. Overall, effort devoted to the first DNA packaging objectives will be more heavily weighted than to the second restriction endonuclease-injected inhibitors portion, the two research objectives of our previous grant. However, approaches to studying DNA translocation in the two research areas will be complementary.