Zhang C, Lee C S, Guo P
Department of Veterinary Pathobiology, Purdue University, West Lafayette, Indiana 47907.
Virology. 1994 May 15;201(1):77-85. doi: 10.1006/viro.1994.1267.
In vitro mutagenesis was performed to identify the DNA packaging domain of the 120-base pRNA essential and specific for DNA encapsidation by bacteriophage phi 29 of Bacillus subtilis. All deletions and mutations targeted the 5' and 3' ends of the pRNA. DNA templates of a control or mutant pRNAs used for in vitro transcription with T7 RNA polymerase were generated by PCR. Fourteen mutant pRNA molecules were synthesized from DNA templates either directly after PCR or after cloning the PCR fragments into the pCR II vector. Ten of the mutant pRNA species were inactive in packaging of the phi 29 genome. Mutation of base one at the 5' end did not affect the pRNA packaging activity. Mutation of the first two bases at the 5' end of the pRNA to noncomplementary bases in the predicted RNA secondary structure (U1 C2/A117G116 to G1 G2/A117G116) resulted in a pRNA with no detectable DNA-gp3 packaging activity assayed by either sucrose gradient sedimentation or agarose gel electrophoresis, and 10(5)-fold reduction in activity was found when measured by plaque-forming units with a new highly sensitive assay system. Changing bases 116 and 117 so that they were complementary to the mutated bases, 1 and 2, from the previous mutant (G1 G2/A117G116 to G1 G2/C117C116) generated an RNA molecule with restored DNA packaging ability. Our results show that, although not essential for procapsid binding, both the 5' and 3' ends of the pRNA were proximate and crucial for phi 29 DNA packaging.
进行了体外诱变，以鉴定对枯草芽孢杆菌噬菌体 phi 29 的 DNA 衣壳化至关重要且特异的 120 个碱基的 pRNA 的 DNA 包装结构域。所有缺失和突变均针对 pRNA 的 5' 和 3' 末端。用于用 T7 RNA 聚合酶进行体外转录的对照或突变 pRNA 的 DNA 模板通过 PCR 产生。从 DNA 模板直接在 PCR 后或在将 PCR 片段克隆到 pCR II 载体后合成了 14 个突变 pRNA 分子。其中 10 种突变 pRNA 物种在 phi 29 基因组的包装中无活性。5' 端第一个碱基的突变不影响 pRNA 的包装活性。将 pRNA 5' 端的前两个碱基突变为预测 RNA 二级结构中的非互补碱基（U1 C2/A117G116 变为 G1 G2/A117G116），通过蔗糖梯度沉降或琼脂糖凝胶电泳检测，得到的 pRNA 没有可检测到的 DNA-gp3 包装活性，并且在使用新的高灵敏度检测系统通过噬斑形成单位测量时，活性降低了 10^5 倍。将碱基 116 和 117 改变为与先前突变体的突变碱基 1 和 2 互补（G1 G2/A117G116 变为 G1 G2/C117C116），产生了具有恢复的 DNA 包装能力的 RNA 分子。我们的结果表明，虽然 pRNA 的 5' 和 3' 末端对于原衣壳结合不是必需的，但它们彼此靠近且对 phi 29 DNA 包装至关重要。
