EARLY EVENTS IN DNA PACKAGING IN A DEFINED INVITRO SYSTEM OF BACTERIOPHAGE-T3
EARLY EVENTS IN DNA PACKAGING IN A DEFINED INVITRO SYSTEM OF BACTERIOPHAGE-T3
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DOI：
10.1016/0042-6822(87)90462-4
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发表时间：
1987-08-01
期刊：
影响因子：
3.7
通讯作者：
MINAGAWA, T
中科院分区：
文献类型：
--
作者：
SHIBATA, H;FUJISAWA, H;MINAGAWA, T
我们已经开发了一种体外包装噬菌体T3 DNA的系统，该系统由纯化的前体蛋白和基因18和19的产物非衣壳蛋白gp18和gp19组成。该反应需要Mg2+、ATP和聚乙二醇，并被一种不可水解的ATP类似物腺苷-5′- o -（3′-硫代三磷酸）（ATP-. γ）抑制。-S) （K. Hamada， H. Fujisawa和T. Minagawa, 1986，病毒学151,119-123）。在定义的系统中，大约30%添加的成熟T3 DNA被包装成头部。沉淀系数约为50s的络合物（50s络合物）在含有ATP- γ -S的反应混合物中积累。50s复合体对DNA酶敏感，在ATP存在的情况下，在不添加DNA、前体、gp18和gp19的情况下，通过第二次反应转化为填充的头部。这些结果表明，在DNA包装的早期阶段，前体复合物的形成是通过ATP作为效应器的变构机制进行的。DNA进入头部的运动是由ATP水解释放的能量驱动的。gp18在不添加ATP- γ的情况下与DNA形成复合物。-S和gp19。gp18-DNA复合物对dna酶敏感，不结合gp19；在加入ATP、gp19和前体后，通过二次反应将其转化为填充的头部。gp19在ATP- γ存在下与prohead形成功能复合物。-S或ATP。该复合物不结合gp18，但通过ATP、gp18和DNA孵育转化为填充头。在没有ATP的情况下。-S， gp19与前体形成复合物，在DNA包装中失败。在ATP- γ -S存在的情况下，gp18-DNA和gp19-prohead复合物混合反应形成50s复合物。根据这些结果，我们提出了在体外系统中定义DNA包装的分子机制的细节。
We have developed a defined in vitro system for packaging phage T3 DNA which is composed of purified proheads and the noncapsid proteins gp18 and gp19, products of genes 18 and 19. The reaction requires Mg2+, ATP, and polyethylene glycol and is inhibited by a nonhydrolyzable ATP analog, adenosine-5''-O-(3''-thiotriphosphate) (ATP-.gamma.-S) (K. Hamada, H. Fujisawa, and T. Minagawa, 1986, Virology 151, 119-123). About 30% of added mature T3 DNA was packaged into heads in the defined system. A complex with a sedimentation coefficient of about 50 S (50 S complex) accumulated in the reaction mixture containing ATP-.gamma.-S. The 50 S complex was DNase sensitive and was converted to filled heads by a second reaction in the presence of ATP without addition of DNA, proheads, gp18, and gp19. These results indicate that during early stages of DNA packaging, formation of precursor complexes proceeds by an allosteric mechanism with ATP acting as effector. The movement of DNA into the head is driven by the energy released by hydrolysis of ATP. gp18 formed a complex with DNA without addition of ATP-.gamma.-S and gp19. gp18-DNA complex was DNase sensitive and did not bind gp19; it was converted to filled heads by way of a second reaction after addition of ATP, gp19, and proheads. gp19 formed a functional complex with prohead in the presence of ATP-.gamma.-S or ATP. The complex did not bind gp18 but was converted to filled heads by incubation with ATP, gp18, and DNA. In the absence of ATP-.gamma.-S, gp19 formed complexes with prohead that were abortive in DNA packaging. Formation of the 50 S complex occurred in a reaction mixture containing gp18-DNA and gp19-prohead complexes in the presence of ATP-.gamma.-S. From these results, we propose details of the molecular mechanism of DNA packaging in the defined in vitro system.