Miller E S, Karam J, Dawson M, Trojanowska M, Gauss P, Gold L
J Mol Biol. 1987 Apr 5;194(3):397-410. doi: 10.1016/0022-2836(87)90670-x.
The RegA protein of bacteriophage T4 is a translational repressor that regulates expression of several phage early mRNAs. We have cloned wild-type and mutant alleles of the T4 regA gene under control of the heat-inducible, plasmid-borne leftward promoter (PL) of phage lambda. Expression of the cloned regA+ gene resulted in the synthesis of a protein that closely resembled phage-encoded RegA protein in biological properties. It repressed its own synthesis (autogenous translational control) as well as the synthesis of specific T4-encoded proteins that are known from other studies to be under RegA-mediated translational control. Cloned mutant alleles of regA exhibited derepressed synthesis of the mutant regA gene products and were ineffective in trans against RegA-sensitive mRNA targets. The effects of plasmid-encoded RegA proteins were also demonstrated in experiments using two compatible plasmids in uninfected Escherichia coli. The two-plasmid assays confirm the sensitivities of several cloned T4 genes to RegA-mediated translational repression and are well-suited for genetic analysis of RegA target sites. Repression specificity in this system was demonstrated by using wild-type and operator-constitutive translational initiation sites of T4 rIIB fused to lacZ. The results show that no additional T4 products are required for RegA-mediated translational repression. Additional evidence is provided for the proposal that uridine-rich mRNA sequences are preferred targets for the repressor. Surprisingly, plasmid-generated RegA protein represses the synthesis of some E. coli proteins and appears to enhance selectively the synthesis of others. The RegA protein may have multiple functions, and its binding sites are not restricted to phage mRNAs.
噬菌体T4的RegA蛋白是一种翻译阻遏物，可调节几种噬菌体早期mRNA的表达。我们已将T4 regA基因的野生型和突变等位基因克隆到受噬菌体λ热诱导型、质粒携带的向左启动子（PL）控制之下。克隆的regA+基因的表达导致一种蛋白质的合成，该蛋白质在生物学特性上与噬菌体编码的RegA蛋白非常相似。它抑制自身的合成（自体翻译控制）以及特定T4编码蛋白的合成，这些蛋白在其他研究中已知受RegA介导的翻译控制。克隆的regA突变等位基因表现出突变regA基因产物的去阻遏合成，并且对RegA敏感的mRNA靶标在反式作用中无效。在未感染的大肠杆菌中使用两个相容质粒的实验中也证明了质粒编码的RegA蛋白的作用。双质粒测定证实了几个克隆的T4基因对RegA介导的翻译阻遏的敏感性，并且非常适合用于RegA靶位点的遗传分析。通过使用与lacZ融合的T4 rIIB的野生型和操纵子组成型翻译起始位点，证明了该系统中的阻遏特异性。结果表明，RegA介导的翻译阻遏不需要额外的T4产物。对于富含尿苷的mRNA序列是阻遏物的优选靶标的提议提供了额外的证据。令人惊讶的是，质粒产生的RegA蛋白抑制一些大肠杆菌蛋白的合成，并且似乎选择性地增强其他蛋白的合成。RegA蛋白可能具有多种功能，并且其结合位点不限于噬菌体mRNA。
