Osawa T, Yura T
Mol Gen Genet. 1980;180(2):293-300. doi: 10.1007/BF00425841.
Amber mutants of Escherichia coli K-12 affected in the structural gene (rpoD) for the sigma subunit of RNA polymerase have been obtained from a strain harboring a temperature-sensitive amber suppressor (supF-Ts6) which is active only at low temperatures. These mutants grow normally at low temperature (30 degrees C) but do not grow at high temperature (42 degrees C) due to the inability to synthesize sigma factor. In one mutant studied in detail (rpoD40), the rate of sigma-factor synthesis at 30 degrees C is about half that of the wild type and is decreased to 10%-15% within 1 h of incubation at 42 degrees C. The synthesis of core polymerase subunits or bulk protein is virtually unaffected at least for 2 h. The defect of the mutant in sigma synthesis and growth at high temperature can be suppressed by any of the amber suppressors tested (supD. supE or supF). RNA-polymerase holoenzymes prepared from the mutant cells carrying each of the suppressors (grown at 42 degrees C) exhibit different thermostabilities attributable to alterations in the sigma factor. The reduced sigma synthesis in the mutant is accompanied by the synthesis of polypeptide tentatively identified as 'amber fragment'. These results as well as the genetic mapping data indicate that the amber mutation (rpoD40) resides within the structural gene for the sigma factor and directly affects sigma synthesis upon inactivation of the suppressor at high temperature.
从携带仅在低温下有活性的温度敏感型琥珀抑制基因（supF-Ts6）的大肠杆菌K-12菌株中，获得了RNA聚合酶σ亚基结构基因（rpoD）发生琥珀突变的突变体。这些突变体在低温（30℃）下能正常生长，但由于无法合成σ因子，在高温（42℃）下不能生长。在一个经过详细研究的突变体（rpoD40）中，30℃时σ因子的合成速率约为野生型的一半，在42℃孵育1小时内降至10%-15%。核心聚合酶亚基或总体蛋白的合成至少在2小时内基本不受影响。该突变体在高温下σ合成和生长的缺陷可被所测试的任何一种琥珀抑制基因（supD、supE或supF）抑制。从携带每种抑制基因（在42℃生长）的突变体细胞中制备的RNA聚合酶全酶，由于σ因子的改变而表现出不同的热稳定性。突变体中σ合成的减少伴随着暂时鉴定为“琥珀片段”的多肽的合成。这些结果以及基因定位数据表明，琥珀突变（rpoD40）位于σ因子的结构基因内，在高温下抑制基因失活时直接影响σ的合成。
