Seong Ki Moon, Park Hweon, Kim Seong Jung, Ha Hyo Nam, Lee Jae Yung, Kim Joon
Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
J Microbiol Methods. 2007 Jun;69(3):442-50. doi: 10.1016/j.mimet.2007.02.010. Epub 2007 Feb 28.
A yeast transcriptional activator, Gcn4p, induces the expression of genes that are involved in amino acid and purine biosynthetic pathways under amino acid starvation. Gcn4p has an acidic activation domain in the central region and a bZIP domain in the C-terminus that is divided into the DNA-binding motif and dimerization leucine zipper motif. In order to identify amino acids in the DNA-binding motif of Gcn4p which are involved in transcriptional activation, we constructed mutant libraries in the DNA-binding motif through an innovative application of random mutagenesis. Mutant library made by oligonucleotides which were mutated randomly using the Poisson distribution showed that the actual mutation frequency was in good agreement with expected values. This method could save the time and effort to create a mutant library with a predictable mutation frequency. Based on the studies using the mutant libraries constructed by the new method, the specific residues of the DNA-binding domain in Gcn4p appear to be involved in the transcriptional activities on a conserved binding site.
酵母转录激活因子Gcn4p在氨基酸饥饿条件下可诱导参与氨基酸和嘌呤生物合成途径的基因表达。Gcn4p在中央区域有一个酸性激活结构域，在C端有一个bZIP结构域，该结构域又分为DNA结合基序和二聚化亮氨酸拉链基序。为了鉴定Gcn4p的DNA结合基序中参与转录激活的氨基酸，我们通过随机诱变的创新应用在DNA结合基序中构建了突变文库。利用泊松分布随机突变的寡核苷酸构建的突变文库表明，实际突变频率与预期值高度一致。该方法可以节省时间和精力来创建具有可预测突变频率的突变文库。基于使用新方法构建的突变文库的研究，Gcn4p中DNA结合结构域的特定残基似乎参与了在保守结合位点上的转录活性。
