Liang Y M, Wei R X, Hsu T, Alford C, Dawson M, Karam J
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425.
Genetics. 1988 Aug;119(4):743-9. doi: 10.1093/genetics/119.4.743.
The regA gene of phage T4 encodes a translational repressor that inhibits utilization of its own mRNA as well as the translation of a number of other phage-induced mRNAs. In recombinant plasmids, autogenous translational repression limits production of the RegA protein when the cloned structural gene is expressed under control of a strong, plasmid-borne promoter (lambda PL). We have found that a genetic fusion which places the regA ribosome binding domain in proximity to active translation leads to partial derepression of wild-type RegA protein synthesis. The derepression is not due to increased synthesis of regA RNA, suggesting that it occurs at the translational level. Derepressed clones of the wild-type regA gene were used to overproduce and purify the repressor. In an in vitro assay the wild-type target was sensitive and a mutant target was resistant to inhibition by the added protein. The results suggest that the sensitivity of a regA-regulated cistron to translational repression may depend on the competition between ribosomes and RegA protein for overlapping recognition sequences in the translation initiation domain of the mRNA.
噬菌体T4的regA基因编码一种翻译阻遏物，它既能抑制自身mRNA的利用，也能抑制许多其他噬菌体诱导的mRNA的翻译。在重组质粒中，当克隆的结构基因在强的、质粒携带的启动子（λPL）控制下表达时，自体翻译阻遏会限制RegA蛋白的产生。我们发现，一种将regA核糖体结合结构域置于接近活性翻译位置的基因融合会导致野生型RegA蛋白合成的部分去阻遏。这种去阻遏不是由于regA RNA合成增加所致，这表明它发生在翻译水平。野生型regA基因的去阻遏克隆用于过量生产和纯化阻遏物。在体外试验中，野生型靶标敏感，而突变型靶标对添加的蛋白抑制具有抗性。结果表明，regA调控的顺反子对翻译阻遏的敏感性可能取决于核糖体与RegA蛋白在mRNA翻译起始结构域中重叠识别序列的竞争。
