Shafranski P, Zagorski V, Khrobochek Ia, zagorska P
Mol Biol (Mosk). 1975 Jan-Feb;9(1):78-85.
One of the mechanisms underlying the regulation of the bacteriophage f2 RNA translation is the repression of the phage RNA-replicase formation by coat protein. This repression is due to the formation of a complex between f2 RNA and coat protein (complex I). In this work the mechanism of complex I formation as well as the effect of this complex on the f2 RNA-replicase formation was followed by inhibition of alanine incorporation into RNA-replicase polypeptide which was separated by polyacrylamide gel electrophoresis. The molar ratios of protein to f2 RNA in complex I were analyzed by sucrose gradient sedimentation. It was been found that complex I consists of six molecules of coat protein bound per one molecule of RNA. Ribonuclease digestion of the glutaraldehyde-fixed complex resulted in a mixture of products in which the hexamers of coat protein molecules were predominant. This indicates that the six molecules of coat protein bound to f2 RNA are neighbouring. It has been also shown that under conditions required for phage protein synthesis, coat protein occurs in solution is dimer. The results show that the translational repression of the RNA-replicase cistron is due to the cooperative attachment of three dimers of coat protein to phage template, forming a hexameric cluster on the RNA strand. The proposed mechanism of the complex I formation seems to be in good agreement with the sequence of events in the phage F2 life cycle. It is known that shortly after infection of the host cell the coat protein and phage RNA-replicase begin to be synthesised. According to our findings, the first portions of coat protein do not affect the translation of the RNA-replicase gene since at low concentration the coat protein occure in the form of monomers. At a later period of phage development, when the concentration of coat protein is sufficiently high to promote the formation of protein dimers, the translational repressor complex is formed and the RNA-replicase gene becomes inoperative.
噬菌体f2 RNA翻译调控的潜在机制之一是外壳蛋白对噬菌体RNA复制酶形成的抑制作用。这种抑制作用是由于f2 RNA与外壳蛋白之间形成了复合物（复合物I）。在这项研究中，通过抑制丙氨酸掺入经聚丙烯酰胺凝胶电泳分离的RNA复制酶多肽中，追踪了复合物I的形成机制以及该复合物对f2 RNA复制酶形成的影响。通过蔗糖梯度沉降分析了复合物I中蛋白质与f2 RNA的摩尔比。结果发现，复合物I由每一个RNA分子结合六个外壳蛋白分子组成。对戊二醛固定的复合物进行核糖核酸酶消化，得到的产物混合物中，外壳蛋白分子的六聚体占主导。这表明与f2 RNA结合的六个外壳蛋白分子是相邻的。还表明，在噬菌体蛋白质合成所需的条件下，溶液中的外壳蛋白以二聚体形式存在。结果表明，RNA复制酶顺反子的翻译抑制是由于三个外壳蛋白二聚体协同附着在噬菌体模板上，在RNA链上形成一个六聚体簇。所提出的复合物I形成机制似乎与噬菌体F2生命周期中的事件顺序高度吻合。已知在宿主细胞感染后不久，外壳蛋白和噬菌体RNA复制酶开始合成。根据我们的发现，外壳蛋白的最初部分不会影响RNA复制酶基因的翻译，因为在低浓度下，外壳蛋白以单体形式存在。在噬菌体发育的后期，当外壳蛋白的浓度足够高以促进蛋白质二聚体的形成时，翻译阻遏复合物形成，RNA复制酶基因失活。
