Ohsawa H, Herrlich P, Gualerzi C
Mol Gen Genet. 1984;196(1):53-8. doi: 10.1007/BF00334091.
Bacteriophage T7 0.3 mRNA synthesised and processed in vitro has been purified starting from the DNA of T7+ as well as from that of two initiation mutants of T7 (CR17 with a U----C transition in the initiation codon and CR35b whose potential Shine and Dalgarno (S-D) interaction is interrupted by a G----A transition). These mRNAs were used as templates to direct the binding of fMet-tRNA and the synthesis of 0.3 protein in both E. coli and wheat germ cell-free systems. The initiation codon mutant displayed approximately 50% inhibition of fMet-tRNA binding and 0.3 protein synthesis in both systems. The S-D sequence mutant, on the other hand, was found to be less affected than the initiation triplet mutant (20%-40% inhibition) in both fMet-tRNA binding and template activity in the E. coli system. In the wheat germ system, which does not make use of the S-D interaction, however, this mutant displayed normal template activity suggesting that the inhibition obtained in the E. coli system, albeit slight, is due to the impairment of the S-D interaction and not to an alteration of the mRNA secondary or tertiary structure caused by the base substitution.
从T7⁺的DNA以及T7的两个起始突变体（起始密码子处有U→C转换的CR17和其潜在的Shine-Dalgarno（S-D）相互作用被G→A转换打断的CR35b）的DNA开始，对体外合成和加工的噬菌体T7 0.3 mRNA进行了纯化。这些mRNA被用作模板，以指导甲硫氨酸起始tRNA的结合以及在大肠杆菌和麦胚无细胞系统中0.3蛋白的合成。起始密码子突变体在两个系统中均表现出约50%的甲硫氨酸起始tRNA结合抑制和0.3蛋白合成抑制。另一方面，在大肠杆菌系统中，发现S-D序列突变体在甲硫氨酸起始tRNA结合和模板活性方面比起始三联体突变体受影响小（20%-40%抑制）。然而，在不利用S-D相互作用的麦胚系统中，该突变体表现出正常的模板活性，这表明在大肠杆菌系统中获得的抑制作用，尽管很轻微，但归因于S-D相互作用的损害，而不是由碱基替换引起的mRNA二级或三级结构的改变。
