Burger G, Strauss J, Scazzocchio C, Lang B F
Institut de Microbiologie, Université de Paris-Sud, Orsay, France.
Mol Cell Biol. 1991 Nov;11(11):5746-55. doi: 10.1128/mcb.11.11.5746-5755.1991.
The nucleotide sequence of nirA, mediating nitrate induction in Aspergillus nidulans, has been determined. Alignment of the cDNA and the genomic DNA sequence indicates that the gene contains four introns and encodes a protein of 892 amino acids. The deduced NIRA protein displays all characteristics of a transcriptional activator. A putative double-stranded DNA-binding domain in the amino-terminal part comprises six cysteine residues, characteristic for the GAL4 family of zinc finger proteins. An amino-terminal highly acidic region and two proline-rich regions are also present. The nucleotide sequences of two mutations were determined after they were mapped by transformation with overlapping DNA fragments, amplified by the polymerase chain reaction. nirA87, a mutation conferring noninducibility by nitrate and nitrite, has a -1 frameshift at triplet 340, which eliminates 549 C-terminal amino acids from the polypeptide. Under the assumption that the truncated polypeptide is stable, it comprises the zinc finger domain and the acidic region, which seem not sufficient for transcriptional activation. nirAd-106, an allele conferring nitrogen metabolite derepression of nitrate and nitrite reductase activity, includes two transitions, changing a glutamic acid to a lysine and a valine to an alanine, situated between a basic and a proline-rich region of the protein. Northern (RNA) analysis of the wild type and of constitutive (nirAc) and derepressed (nirAd) mutants show that the nirA transcript does not vary between these strains, being in all cases constitutively expressed. On the other hand, transcript levels of structural genes (niaD and niiA) do vary, being highly inducible in the wild type but constitutively expressed in the nirAc mutant. The nirAd mutant appears phenotypically derepressed, because the niaD and niiA transcript levels are overinduced in the presence of nitrate but are still partially repressed in the presence of ammonium.
已确定在构巢曲霉中介导硝酸盐诱导作用的nirA的核苷酸序列。cDNA与基因组DNA序列的比对表明，该基因含有四个内含子，编码一个由892个氨基酸组成的蛋白质。推导的NIRA蛋白具有转录激活因子的所有特征。氨基末端部分的一个假定的双链DNA结合结构域包含六个半胱氨酸残基，这是GAL4家族锌指蛋白的特征。还存在一个氨基末端高度酸性区域和两个富含脯氨酸的区域。通过用重叠DNA片段转化进行定位后，确定了两个突变的核苷酸序列，这些片段通过聚合酶链反应进行扩增。nirA87是一种导致对硝酸盐和亚硝酸盐无诱导性的突变，在三联体340处有一个-1移码，从多肽中消除了549个C末端氨基酸。假设截短的多肽是稳定的，它包含锌指结构域和酸性区域，这似乎不足以进行转录激活。nirAd-106是一个赋予硝酸盐和亚硝酸盐还原酶活性氮代谢物去阻遏作用的等位基因，包括两个转换，将一个谷氨酸变为赖氨酸，一个缬氨酸变为丙氨酸，位于该蛋白质的一个碱性区域和一个富含脯氨酸的区域之间。对野生型、组成型（nirAc）和去阻遏型（nirAd）突变体进行的Northern（RNA）分析表明，nirA转录本在这些菌株之间没有变化，在所有情况下都是组成型表达。另一方面，结构基因（niaD和niiA）的转录本水平确实有所不同，在野生型中高度可诱导，但在nirAc突变体中组成型表达。nirAd突变体在表型上似乎是去阻遏的，因为在有硝酸盐存在时niaD和niiA转录本水平过度诱导，但在有铵存在时仍部分受到抑制。
