RNA-BINDING, PACKAGING AND POLYMERASE ACTIVITIES OF THE DIFFERENT INCOMPLETE POLYMERASE COMPLEX PARTICLES OF DSRNA BACTERIOPHAGE-PHI-6
RNA-BINDING, PACKAGING AND POLYMERASE ACTIVITIES OF THE DIFFERENT INCOMPLETE POLYMERASE COMPLEX PARTICLES OF DSRNA BACTERIOPHAGE-PHI-6
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DOI：
10.1006/jmbi.1995.0317
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发表时间：
1995-06-09
影响因子：
5.6
通讯作者：
BAMFORD, DH
中科院分区：
文献类型：
--
作者：
JUUTI, JT;BAMFORD, DH
phi 6是一种有包膜的dsRNA细菌病毒。其分段基因组位于病毒体相关聚合酶复合体内，该复合体由病毒L片段编码的四种蛋白质（P1、P2、P4和P7）形成。完整和不完整的聚合酶复合物颗粒可以使用该最大基因组区段的cDNA拷贝产生。我们分析了不同纯化颗粒（1）包装phi 6（+）有义基因组前体和非特异性RNA，（2）合成（-）和（+）链和（3）结合phi 6特异性和非特异性RNA的能力。发现（-）和（+）链合成聚合酶活性与蛋白P2相关。除了完整的颗粒外，发现缺乏蛋白P2的颗粒包装和保护基因组前体ssRNA。P7蛋白是高效包装所必需的。包装的调节和特异性被认为是独立的P2。由蛋白质P1和P4组成的颗粒不包装或保护RNA，但结合phi 6基因组（+）链RNA。当在过滤器结合测定中单独测试时，三条phi 6（+）链以等量结合颗粒。在竞争实验中，它们相互竞争结合，表明三条基因组（+）链的单个结合位点不存在。观察到四种颗粒之间RNA结合竞争的差异，表明包装特异性是通过在初始结合事件后包装过程的推进期间蛋白质和基因组（+）链RNA的复杂相互作用实现的。
phi 6 is an enveloped dsRNA bacterial virus. Its segmented genome resides inside the virion associated polymerase complex which is formed by four proteins (P1, P2, P4 and P7) encoded by the viral L segment. Complete and incomplete polymerase complex particles can be produced using cDNA copies of this largest genome segment. We have analysed the capacity of the different purified particles to (1) package phi 6 (+) sense genomic precursors and unspecific RNA, (2) synthesize (-) and (+) strands and (3) bind phi 6 specific and unspecific RNAs. Both (-) and (+) strand synthesis polymerase activities were found to be associated with protein P2. In addition to complete particles, particles lacking protein P2 were found to package and protect genomic precursor ssRNAs. Protein P7 was needed for efficient packaging. Regulation and specificity of the packaging were found to be independent of P2. Particles composed of proteins P1 and P4 did not package or protect RNA but did bind phi 6 genomic (+) strand RNAs. The three phi 6 (+) strands bound in equal amounts to the particles when tested alone in a filter binding assay. In competition experiments they competed each other for binding, indicating that individual binding sites for the three genomic (+) strands do not exist. Differences in RNA binding competition among the four particles were observed, suggesting that packaging specificity is achieved by complex interactions of proteins and genomic (+) strand RNAs during the advancement of the packaging process after the initial binding events.