Phage T4 Head Assembly and Initiation of Infection

描述（由申请人提供）：资助申请提出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节; 2）注射以对抗新型DNA易位限制酶的内部蛋白质的进化。包装马达由T4大末端酶亚基组成，其在前体的门十二聚体处工作，以接近100%的效率转运染料标记的任何序列的短DNA。在单个复合物之后，我们将分析门户-GFP prohead包装。通过FCS和核酸酶测定，短的切口DNA不被包装，不像气泡错配DNA，这表明马达将扭转引入DNA中以使其移位。FRET将用于用双染料DNA确定DNA的尺寸被失速的马达改变。此外，在标记的末端酶和门户之间和之内的FRET将在包装期间建立距离和构象变化。FRET还将用于确定包装头部两端的接近度。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，显示晚西格玛因子（gp 55）-滑动钳（gp 45）复合物的参与，在包装启动在体内的缺口环状DNA的大末端酶亚基gp 17与一些调控蛋白的相互作用。在第二个目标中，通过在IPI基因座的基因扩增进化了一个不同的CTS注射蛋白家族，以挑战NTP驱动的DNA易位限制酶的新家族。我们正在进行的两个gmrS/gmrD限制性内切核酸酶（CT和UT），两个多态性噬菌体基因拮抗剂（IPI* 和IP 5 *，其结构，我们已经确定），和不同的目标糖HMC加合物，这些酶的分析揭示了一个进化途径产生的纳米级的复杂性。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核酸内切酶注射抑制剂部分更重要，这是我们以前资助的两个研究目标。然而，在这两个研究领域研究DNA易位的方法将是互补的。

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.