Hu D S, Hood D W, Heidstra R, Hodgson D A
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Mol Microbiol. 1999 May;32(4):869-80. doi: 10.1046/j.1365-2958.1999.01407.x.
Transformation of tryptophan auxotrophs of Streptomyces coelicolor A3(2) and subsequent analysis have allowed the identification of four tryptophan biosynthetic genes. Subcloning, complementation of trp strains, nucleotide sequencing of 5.1 kb and 1.95 kb of DNA and subsequent homology comparisons identified the trpC, trpB and trpA genes and trpD gene respectively. The arrangement of genes in the trpCBA cluster is unusual in that trpC is separated by a small open reading frame, trpX, from the potentially translationally coupled trpB and trpA genes. Sequence analysis of the trpD gene revealed the presence of a large mRNA loop structure directly upstream of the trpD-coding region. S1 nuclease mapping studies of trpCXBA have revealed two major potential transcription start points, one just upstream of the trpC gene and the other located upstream of the trpX gene. S1 nuclease mapping of the trpD region revealed four fragment end-points. Quantitative S1 nuclease protection assays and a promoterless catechol dioxygenase reporter gene have revealed that the expression of all these genes is growth phase dependent and growth rate dependent, expression being maximal during early exponential phase and dropping off sharply in late exponential phase. This growth phase-dependent and growth rate-dependent regulation is the first reported in streptomycete primary metabolism.
对天蓝色链霉菌A3(2)的色氨酸营养缺陷型进行转化并随后进行分析,已鉴定出四个色氨酸生物合成基因。通过亚克隆、色氨酸菌株的互补、对5.1 kb和1.95 kb DNA的核苷酸测序以及随后的同源性比较,分别鉴定出了trpC、trpB和trpA基因以及trpD基因。trpCBA基因簇中的基因排列方式不同寻常,因为trpC与可能翻译偶联的trpB和trpA基因被一个小的开放阅读框trpX隔开。trpD基因的序列分析显示,在trpD编码区的直接上游存在一个大的mRNA环结构。对trpCXBA的S1核酸酶图谱研究揭示了两个主要的潜在转录起始点,一个在trpC基因的上游,另一个位于trpX基因的上游。trpD区域的S1核酸酶图谱显示有四个片段端点。定量S1核酸酶保护分析和一个无启动子的儿茶酚双加氧酶报告基因显示,所有这些基因的表达都依赖于生长阶段和生长速率,在指数早期阶段表达最高,在指数后期阶段急剧下降。这种依赖于生长阶段和生长速率的调控是链霉菌初级代谢中首次报道的。