Schreier H J, Rostkowski C A
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore 21202, USA.
Gene. 1995 Aug 8;161(1):51-6. doi: 10.1016/0378-1119(95)00238-2.
The Bacillus subtilis glnR gene (part of the glnRA operon) encodes a 135-amino-acid (aa) repressor, GlnR, that regulates glnRA transcription in response to nitrogen levels in the growth medium. Two glnR mutants unable to repress under nitrogen excess conditions were obtained by mutagenesis. Lesions were found at Leu77 and Ala80, aa that lie within a region (between aa 59-83) thought to form the alpha-helix-turn-alpha-helix (HTH) motif common among a class of regulatory proteins. Alteration of Gly72 by site-directed mutagenesis also affected regulation, suggesting that aa within the putative HTH region are critical for GlnR function and may be involved in DNA binding. However, other replacements within the aa 59-83 sequence failed to support the HTH structure proposed for this region. Mutations within the C-terminal region of GlnR were also found to affect regulation. Introduction of an ochre stop codon at aa 110, 116, 123 and 129 resulted in the production of truncated proteins that were constitutively repressed, strongly suggesting that a signal recognition site residues within the last seven aa of GlnR. Substituting Asp129 with Asn led to loss of repression, indicating that Asp129 may be directly involved in interacting with either positive or negative effector molecules, or is a target for post-translational modification.
枯草芽孢杆菌的谷氨酰胺调节基因（谷氨酰胺调节基因操纵子的一部分）编码一种含135个氨基酸的阻遏蛋白GlnR，该蛋白可根据生长培养基中的氮水平调节谷氨酰胺调节基因的转录。通过诱变获得了两个在氮过量条件下无法进行阻遏的谷氨酰胺调节基因突变体。在第77位亮氨酸和第80位丙氨酸处发现了损伤，这些氨基酸位于一个区域（第59 - 83位氨基酸之间），该区域被认为可形成一类调节蛋白中常见的α - 螺旋 - 转角 - α - 螺旋（HTH）基序。通过定点诱变改变第72位甘氨酸也影响了调节作用，这表明假定的HTH区域内的氨基酸对GlnR功能至关重要，可能参与DNA结合。然而，第59 - 83位氨基酸序列中的其他替换未能支持为该区域提出的HTH结构。还发现GlnR C末端区域内的突变会影响调节作用。在第110、116、123和129位氨基酸处引入赭石色终止密码子导致产生截短的蛋白质，这些蛋白质持续受到阻遏，这强烈表明在GlnR的最后七个氨基酸内存在一个信号识别位点残基。将第129位天冬氨酸替换为天冬酰胺导致阻遏作用丧失，表明天冬氨酸129可能直接参与与正效应分子或负效应分子的相互作用，或者是翻译后修饰的靶点。
