Eisenberg S P, Soll L, Yarus M
J Biol Chem. 1979 Jun 25;254(12):5562-6.
Escherichia coli can be temperature-sensitive due to a lesion in the gene for tRNATrp (Yanofsky, C., and Soll, L. (1977) J. Mol. Biol. 113, 663-677). Purification of tRNATrp from this strain (temperature-sensitive tRNATrp) was achieved by one of two methods. Either a combination of benzoylated DEAE-cellulose column chromatography and two-dimensional polyacrylamide gel electrophoresis, or hybridization to plasmid DNA covalently bound to cellulose (this is a recombinant plasmid carrying the gene for tRNATrp) and electrophoresis of the eluted material on a 10% polyacrylamide gel, produced isotopically pure tRNA. The sequence of the temperature-sensitive tRNATrp was determined by standard methods. We find that the sequence differs from that of wild type tRNATrp by a single residue; G in position 7 (G7) in wild type tRNATrp is A7 in temperature-sensitive tRNATrp. This base difference results in one less base pair in the CCA stem of the temperature-sensitive species. The effect of this base change in the in vitro and in vivo properties of tRNATrp (presented elsewhere (S.P. Eisenberg and M. Yarus, manuscript in preparation.)) are discussed.
由于色氨酸转运RNA（tRNATrp）基因发生损伤，大肠杆菌可能对温度敏感（亚诺夫斯基，C.，和索尔，L.（1977年）《分子生物学杂志》113卷，663 - 677页）。从该菌株（温度敏感型tRNATrp）中纯化tRNATrp可通过两种方法之一实现。要么采用苯甲酰化二乙氨基乙基纤维素柱层析和二维聚丙烯酰胺凝胶电泳相结合的方法，要么与共价结合到纤维素上的质粒DNA（这是一种携带tRNATrp基因的重组质粒）进行杂交，并将洗脱物在10%聚丙烯酰胺凝胶上进行电泳，从而得到同位素纯的tRNA。温度敏感型tRNATrp的序列通过标准方法测定。我们发现该序列与野生型tRNATrp的序列仅在一个残基上存在差异；野生型tRNATrp中第7位的鸟嘌呤（G7）在温度敏感型tRNATrp中为腺嘌呤（A7）。这种碱基差异导致温度敏感型tRNATrp的CCA茎中碱基对比野生型少一对。本文讨论了这种碱基变化对tRNATrp体外和体内特性的影响（相关内容见其他文章（S.P. 艾森伯格和M. 亚鲁斯，正在准备的手稿））。
