Vacher J, Springer M, Buckingham R H
EMBO J. 1985 Feb;4(2):509-13. doi: 10.1002/j.1460-2075.1985.tb03657.x.
The gene pheV from Escherichia coli, coding for tRNAPhe and carried on a plasmid, has been mutagenised with hydroxylamine. Mutants in the structural gene have been identified using two criteria: (i) de-attenuation of beta-galactosidase expression, while under the control of the attenuator region of the pheS,T operon by means of an operon fusion; (ii) loss of ability to complement thermosensitivity of a mutant Phe-tRNA synthetase. Mutants showing de-attenuation were sequenced and two nucleotide changes identified: G44----A44 (found five times) and m7G46----A46 (found once). Sequencing of mutants that lost complementation identified two further tRNA mutants, C2---U2 and G15----A15; the mutant m7G46----A46 was also re-isolated by this criterion. Three of the mutants involve bases implicated in tertiary rather than secondary structure hydrogen bonding. One hypothesis for the mechanism of de-attenuation is that mutant tRNAPhe molecules compete with the wild-type tRNAPhe on the ribosome but are inefficient at some step in the elongation process.
来自大肠杆菌的编码苯丙氨酸tRNA的pheV基因，携带在一个质粒上，已用羟胺进行诱变。利用两个标准鉴定了结构基因中的突变体：（i）在通过操纵子融合受pheS、T操纵子衰减子区域控制时，β-半乳糖苷酶表达的去衰减；（ii）丧失对突变型苯丙氨酸-tRNA合成酶热敏感性的互补能力。对显示去衰减的突变体进行测序，鉴定出两个核苷酸变化：G44→A44（发现5次）和m7G46→A46（发现1次）。对丧失互补作用的突变体进行测序，又鉴定出另外两个tRNA突变体，C2→U2和G15→A15；突变体m7G46→A46也通过这一标准再次分离得到。其中三个突变体涉及与三级结构而非二级结构氢键有关的碱基。去衰减机制的一种假说是，突变型苯丙氨酸tRNA分子在核糖体上与野生型苯丙氨酸tRNA竞争，但在延伸过程的某个步骤效率低下。
