Tu H, Casadaban M J
Department of Molecular Genetics and Cell Biology, University of Chicago, IL 60637.
Nucleic Acids Res. 1990 Jul 11;18(13):3923-31. doi: 10.1093/nar/18.13.3923.
The upstream activating sequence (UAS) conferring leucine-specific regulation of transcription in Saccharomyces cerevisiae was identified by analysis of the LEU2 promoter and by comparison to other genes regulated by leucine. The UAS was localized with deletions and cloned synthetic DNA. Point mutations and sequence rearrangements were used to identify important basepairs and to construct an improved UAS with increased regulation and expression. The improved UAS contains a core ten basepair, GC-rich, palindromic sequence, which is sufficient to confer minimal levels of activation and regulation, within a 36 basepair palindromic sequence which confers maximal activation and regulation. Deletions downstream of the UAS indicated that the UAS must act in conjunction with at least one other site, perhaps a TATAA region, in order to confer high levels of activation. Tandem copies of the UAS in front of LEU2 increased expression and regulation. Tandem UAS elements in trans on a multi-copy 2 mu-based plasmid decreased expression and regulation. These results are consistent with a model that the UAS serves as the DNA-binding site for diffusible activation factor(s), possibly the LEU3 gene product.
通过对LEU2启动子的分析以及与其他受亮氨酸调控的基因进行比较，确定了酿酒酵母中赋予亮氨酸特异性转录调控的上游激活序列（UAS）。利用缺失定位和克隆合成DNA对UAS进行定位。通过点突变和序列重排来确定重要碱基对，并构建了一个调控和表达能力增强的改良型UAS。改良型UAS包含一个由十个碱基对组成的富含GC的核心回文序列，在一个赋予最大激活和调控能力的36个碱基对的回文序列中，该核心序列足以赋予最低水平的激活和调控。UAS下游的缺失表明，UAS必须与至少一个其他位点（可能是TATAA区域）协同作用，才能赋予高水平的激活。LEU2前的UAS串联拷贝增加了表达和调控。多拷贝2μm质粒上反式排列的串联UAS元件降低了表达和调控。这些结果与一个模型一致，即UAS作为可扩散激活因子（可能是LEU3基因产物）的DNA结合位点。
