Rowitch D H, Hunter G J, Perham R N
Department of Biochemistry, University of Cambridge, England.
J Mol Biol. 1988 Dec 5;204(3):663-74. doi: 10.1016/0022-2836(88)90363-4.
A restriction fragment carrying the major coat protein gene (gene VIII) was excised from the DNA of the class I filamentous bacteriophage fd, which infects Escherichia coli. This fragment was cloned into the expression plasmid pKK223-3, where it came under the control of the tac promoter, generating plasmid pKf8P. Bacteriophage fd gene VIII was similarly cloned into the plasmid pEMBL9+, enabling it to be subjected to site-directed mutagenesis. By this means the positively charged lysine residue at position 48, one of four positively charged residues near the C terminus of the protein, was turned into a negatively charged glutamic acid residue. The mutated fd gene VIII was cloned back from the pEMBL plasmid into the expression plasmid pKK223-3, creating plasmid pKE48. In the presence of the inducer isopropyl-beta-D-thiogalactoside, the wild-type and mutated coat protein genes were strongly expressed in E. coli TG1 cells transformed with plasmids pKf8P and pKE48, respectively, and the product procoat proteins underwent processing and insertion into the E. coli cell inner membrane. A net positive charge of only 2 on the side-chains in the C-terminal region is evidently sufficient for this initial stage of the virus assembly process. However, the mutated coat protein could not encapsidate the DNA of bacteriophage R252, an fd bacteriophage carrying an amber mutation in its own gene VIII, when tested on non-suppressor strains of E. coli. On the other hand, elongated hybrid bacteriophage particles could be generated whose capsids contained mixtures of wild-type (K48) and mutant (E48) subunits. This suggests that the defect in assembly may occur at the initiation rather than the elongation step(s) in virus assembly. Other mutations of lysine-48 that removed or reversed the positive charge at this position in the C-terminal region of the coat protein were also found to lead to the production of commensurately longer bacteriophage particles. Taken together, these results indicate direct electrostatic interaction between the DNA and the coat protein in the capsid and support a model of non-specific binding between DNA and coat protein subunits with a stoicheiometry that can be varied during assembly.
从感染大肠杆菌的I类丝状噬菌体fd的DNA中切下携带主要外壳蛋白基因（基因VIII）的限制性片段。该片段被克隆到表达质粒pKK223 - 3中，在tac启动子的控制下，产生质粒pKf8P。噬菌体fd基因VIII同样被克隆到质粒pEMBL9 + 中，使其能够进行定点诱变。通过这种方法，蛋白质C末端附近四个带正电荷的残基之一、位于第48位的带正电荷的赖氨酸残基被转化为带负电荷的谷氨酸残基。突变的fd基因VIII从pEMBL质粒中克隆回表达质粒pKK223 - 3，产生质粒pKE48。在诱导剂异丙基 - β - D - 硫代半乳糖苷存在的情况下，野生型和突变型外壳蛋白基因分别在用质粒pKf8P和pKE48转化的大肠杆菌TG1细胞中强烈表达，并且产物原外壳蛋白经历加工并插入大肠杆菌细胞内膜。C末端区域侧链上仅2的净正电荷显然足以满足病毒组装过程的这一初始阶段。然而，当在大肠杆菌的非抑制菌株上进行测试时，突变的外壳蛋白不能包裹噬菌体R252的DNA，R252是一种在其自身基因VIII中携带琥珀突变的fd噬菌体。另一方面，可以产生细长的杂交噬菌体颗粒，其衣壳包含野生型（K48）和突变型（E48）亚基的混合物。这表明组装缺陷可能发生在病毒组装的起始阶段而不是延伸步骤。还发现外壳蛋白C末端区域中去除或反转该位置正电荷的赖氨酸 - 48的其他突变也导致产生相应更长的噬菌体颗粒。综上所述，这些结果表明衣壳中DNA与外壳蛋白之间存在直接的静电相互作用，并支持DNA与外壳蛋白亚基之间非特异性结合的模型，其化学计量在组装过程中可以变化。
