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Analyses of the Relationship between Amino Acid Substitution and Phenotype of the Tail Sheath Protein of Bacteriophage T4

噬菌体T4尾鞘蛋白氨基酸取代与表型关系分析

基本信息

批准号：
02680125
负责人：
ARISAKA Fumio
金额：
$ 1.28万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02680125/
关键词：
Bacteriophage T4 phage Contractile Tail Sheath Tail Sheath Protein Mutant Temperature Sensitivity Cold Sensitivity Nonsense Mutant 収縮性尾鞘蛋白質 PCR ジデオキシ法アンバ-

项目摘要

Five phenotypes of mutants which have a mutation in gene 18 have been reported, namely amber (am), temperature sensitive (ts : grow at 30ﾟC, but not at 42ﾟC), cold sensitive (cs : grow at 37ﾟC, but not at 25ﾟC), heat labile or sensitive (hs) and polyethylene glycol insensitive (CBW). We collected these mutants from original authors and have identified the mutation sites on gene 18. In order to determine the mutation sites, the mutant gene 18 were amplified by the asymmetric PCR method and directly sequenced by the dideoxy method. Mutation sites of 17 am, 3 cs, 3 ts, 3 CBW and 4 hs mutants have thus been identified. Amber mutations were mapped both in N-terminal and in C-terminal half of the molecule. On the other hand, hs and cs mutations which are considered to be related to inter-subunit interactions were mapped in a rather restricted region in the C-terminal half of the molecule except for one hs mutant which was mapped close to the N-terminus of gp18 molecule. Ts mutations were mapped in the N-terminal half of the molecule. For amber mutants, E. coli natural and artificial suppressor strains were used to insert an amino acid (Ser, Gln, Tyr, Ala, Cys, Glu, His, Phe, Pro or Arg) into each amber site and recovery of the function and its temperature dependance were examined. For missense mutants (cs, ts, hs and CBW), the relationship between each phenotype and the amino acid replacement were reinvestigated. The results were all consistent with our model of the tertiary and quartenary structure of gp18, in which the protease-resistant domain (residue numbers between ca. 80 and 320) constitutes the protruding part of the tail sheath and the N-terminal part (ca. 1-80) and the C-terminal half of the molecule (ca. 320 - 658) constitutes the inner part of the tail sheath in contact with other protomers of gp18 and the tube structure.

已经报道了5种18基因突变的突变体，即琥珀型(Am)、温度敏感型(ts：在30ﾟC生长，但不在42ﾟC生长)、冷敏型(cs：在37ﾟC生长，但不在25ﾟC生长)、热不稳定或敏感(Hs)和聚乙二醇不敏感(Cbw)。我们从原始作者那里收集了这些突变体，并确定了基因18上的突变位点。为了确定突变位点，用不对称聚合酶链式反应方法扩增突变基因18，并用双脱氧法直接测序。从而确定了17am、3cs、3ts、3cbw和4hs突变体的突变位点。琥珀突变在分子的N-末端和C-末端都被定位。另一方面，被认为与亚基间相互作用有关的hs和cs突变被定位在分子的C末端的一个相当有限的区域，除了一个hs突变体被定位在gp18分子的N端附近。TS突变被定位在分子的N-末端半部分。对于琥珀突变体，利用大肠杆菌天然和人工抑制菌株在每个琥珀位点插入一个氨基酸(Ser、Gln、Tyr、Ala、Cys、Glu、His、Phe、Pro或Arg)，并考察其功能恢复及其温度依赖性。对于错义突变体(cs、ts、hs和cbw)，重新研究了每种表型与氨基酸替换的关系。这些结果都与我们的gp18三级和四级结构模型一致，其中蛋白酶抗性结构域(残基数约为80-320)构成了尾鞘的突出部分，N-末端部分(约1-80)和分子的C-末端半部分(约320-658)构成了尾鞘的内侧部分，与gp18的其他原基和管状结构接触。