Site-directed mutagenesis of an amino acid residue in the bacteriophage P2 ogr protein implicated in interaction with Escherichia coli RNA polymerase.
Site-directed mutagenesis of an amino acid residue in the bacteriophage P2 ogr protein implicated in interaction with Escherichia coli RNA polymerase.
复制标题
噬菌体 P2 ogr 蛋白中与大肠杆菌 RNA 聚合酶相互作用有关的氨基酸残基的定点诱变。
DOI：
10.1111/j.1365-2958.1992.tb02199.x
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发表时间：
1992
影响因子：
3.6
通讯作者：
Christie,GE
中科院分区：
文献类型：
--
作者：
King,RA;Anders,DL;Christie,GE
P2基因编码一个72个氨基酸的蛋白质，是P2晚期基因表达所必需的。该基因最初是由一类补偿突变定义的，这种突变克服了宿主突变rpoA109对P2晚期转录的阻碍，该突变编码大肠杆菌RNA聚合酶的a亚基。自发的代偿酸突变取代了OGR多肽中42位氨基酸的酪氨酸残基。利用无序突变抑制和寡核苷酸定点突变的方法，我们研究了OGR中该位置氨基酸替换的影响。该位点上带电残基的替换使OGR蛋白失活，失活的菌株为A+和A109。而11种不同的氨基酸能够取代野生型Tyr-42，以允许野生Typee在不同程度上生长P2。其中只有三种允许携带therpoA109突变的菌株中的噬菌体生长。携带半胱氨酸或丙氨酸取代Tyr-42的噬菌体在arpoA+菌株中的突发性大小至少高达P2ogr+；甘氨酸替代也允许P2在arpoA+或rpoA109背景下生长，但显著减小突发性大小。这些结果与OGR和E的α亚基之间的直接作用是一致的。CoilRNA聚合酶对P2晚期转录的阳性对照，表明热A109突变的阻断是由于空间位阻所致。
The P2ogrgene encodes a 72–amino–acid protein required for P2 late gene expression. This gene was defined originally by a class of compensatory mutations which overcome the block to P2 late transcription imposed by a host mutation,rpoA109, in the gene encoding the a subunit ofEscherichia coliRNA polymerase. Spontaneous compensatoryogrmutations substitute a Cys for a Tyr residue at amino acid 42 in the Ogr polypeptide. Using suppression of anogramber mutation and site–directed oligonucleotide mutagenesis, we have studied the effect of amino acid substitutions at this position in Ogr. Substitution of charged residues at this site renders Ogr protein inactive, inrpoA+andrpoA109strains. While 11 different amino acids are capable of replacing the wild–type Tyr–42 to allow P2 growth to varying degrees in a wild–typeE. colistrain, only three of these allow phage growth in strains carrying therpoA109mutation. Phages carrying Cys or Ala in place of Tyr–42 gave burst sizes at least as high as P2ogr+in arpoA+strain; a Gly substitution also allowed P2 to grow in either arpoA+orrpoA109background, but markedly reduced the burst size. These results are consistent with a direct interaction between Ogr and the α subunit ofE. coilRNA polymerase in positive control of P2 late transcription, and indicate that the block imposed by therpoA109mutation is due to steric hindrance.