Danot O, Vidal-Ingigliardi D, Raibaud O
Unité de Génétique Moléculaire, (CNRS URA 1149), Institut Pasteur, Paris, France.
J Mol Biol. 1996 Sep 13;262(1):1-11. doi: 10.1006/jmbi.1996.0493.
MalT is the transcriptional activator of the Escherichia coli maltose regulon. Several lines of evidence suggest that MalT might act by interacting with RNA polymerase. Here, we show that 'MalT, the DNA-binding domain of MalT, activates transcription. In order to identify amino acids of 'MalT playing a specific role in activation, and therefore possibly involved in the putative contact(s) with RNA polymerase, we developed a double screen to isolate mutations of the 'malT gene affecting activation by 'MalT without impairing its DNA-binding affinity. The effect of the mutations thus obtained on activation was assessed in vivo. This strategy essentially pointed to serine 834 and glutamine 876 of the MalT amino acid sequence as specifically involved in activation. Various 'MalT derivatives substituted at positions 834 or 876 were purified and tested in vitro for their DNA-binding affinity, as well as for their activation ability. Together, the results obtained clearly show that serine 834 and glutamine 876 are important for activation by 'MalT but not for DNA-binding. We argue that these amino acid residues are possibly solvent-exposed and propose that they act by contacting RNA polymerase.
MalT是大肠杆菌麦芽糖操纵子的转录激活因子。多项证据表明，MalT可能通过与RNA聚合酶相互作用来发挥作用。在此，我们表明MalT的DNA结合结构域MalT可激活转录。为了确定MalT中在激活过程中起特定作用、因此可能参与与RNA聚合酶假定接触的氨基酸，我们开发了一种双重筛选方法，以分离影响MalT激活但不损害其DNA结合亲和力的malT基因突变。对由此获得的突变体在体内的激活作用进行了评估。该策略主要指出MalT氨基酸序列中的丝氨酸834和谷氨酰胺876特别参与激活过程。对在834或876位被取代的各种MalT衍生物进行了纯化，并在体外测试了它们的DNA结合亲和力以及激活能力。综合起来，所获得的结果清楚地表明，丝氨酸834和谷氨酰胺876对MalT的激活很重要，但对DNA结合不重要。我们认为这些氨基酸残基可能暴露于溶剂中，并提出它们通过与RNA聚合酶接触来发挥作用。
