Delamarche C, Vacher J, Buckingham R H
Institut de Biologie Physico-Chimique, Paris, France.
Eur J Biochem. 1987 Oct 15;168(2):365-9. doi: 10.1111/j.1432-1033.1987.tb13428.x.
Four mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, share the characteristic that when carried in the plasmid pBR322, they lose the capacity of wild-type pheV to complement the thermosensitive defect in a mutant of phenylalanyl-tRNA synthetase. One of these mutants, leading to the change C2----U2 in tRNA(Phe), is expressed about 10-fold lower in transformed cells than wild-type pheV. This mutant, unlike the remaining three (G15----A15, G44----A44, m7G46----A46), can recover the capacity to complement thermosensitivity when carried in a plasmid of higher copy number. The other three mutants, even when expressed at a similar level, remain unable to complement thermosensitivity. A study of charging kinetics suggests that the loss of complementation associated with these mutants is due to an altered interaction with phenylalanyl-tRNA synthetase. The mutant gene pheV (U2), when carried in pBR322, can also recover the capacity to complement thermosensitivity through a second-site mutation outside the tRNA structural gene, in the discriminator region. This mutation, C(-6)----T(-6), restores expression of the mutant U2 to about the level of wild-type tRNA(Phe).
编码大肠杆菌苯丙氨酰 - tRNA（Phe）的基因pheV的四个突变体具有共同特征，即当它们存在于质粒pBR322中时，会丧失野生型pheV互补苯丙氨酰 - tRNA合成酶突变体中温度敏感缺陷的能力。其中一个突变体导致tRNA（Phe）中的C2----U2变化，在转化细胞中的表达比野生型pheV低约10倍。与其余三个突变体（G15----A15、G44----A44、m7G46----A46）不同，这个突变体当存在于高拷贝数质粒中时能够恢复互补温度敏感性的能力。其他三个突变体，即使在相似水平表达时，仍然无法互补温度敏感性。对充电动力学的研究表明，与这些突变体相关的互补能力丧失是由于与苯丙氨酰 - tRNA合成酶的相互作用改变所致。当突变基因pheV（U2）存在于pBR322中时，也能够通过tRNA结构基因外鉴别区域的第二位点突变恢复互补温度敏感性的能力。这个突变，C（-6）----T（-6），将突变体U2的表达恢复到大约野生型tRNA（Phe）的水平。
