Ooi B G, Rankin C, Miller L K
Department of Entomology, University of Georgia, Athens 30602.
J Mol Biol. 1989 Dec 20;210(4):721-36. doi: 10.1016/0022-2836(89)90105-8.
A series of recombinant baculoviruses containing linker-substituted polyhedrin promoters attached to a reporter gene encoding chloramphenicol acetyl transferase (CAT) were constructed and tested for expression of the gene. The major determinant for promoter activity was narrowed to within eight nucleotides, TAAGTATT, at the start point of polyhedrin mRNA transcription. Mutations within TAAGTATT blocked initiation of transcription from this site and resulted in a 2000-fold decrease in CAT activity. Linker mutations from 12 to 22 bases upstream from the TAAGTATT sequence increased the steady-state levels of RNAs initiated within TAAGTATT and increased CAT expression by up to 50%. Mutations downstream from TAAGTATT and within the region specifying the untranslated RNA leader diminished transcriptional initiation at TAAGTATT and decreased CAT activity two- to 20-fold. The half-lives of CAT RNAs were not noticeably affected by mutations in the untranslated RNA leader region and thus RNA turn-over was not responsible for the reduced levels of these CAT RNAs. Nuclear run-on analysis of two mutant viruses showed that these mutations decrease the rate of transcriptional initiation. Transcriptional initiation thus appears to be the major means of polyhedrin gene regulation. The data define promoter-related roles for TAAGTATT and the sequences specifying the untranslated mRNA leader in transcriptional initiation. A model by which the viral-induced RNA polymerase distinguishes late and very late initiation sites is proposed.
构建了一系列含有与编码氯霉素乙酰转移酶（CAT）的报告基因相连的接头取代多角体蛋白启动子的重组杆状病毒，并对该基因的表达进行了检测。多角体蛋白mRNA转录起始点处启动子活性的主要决定因素被缩小到八个核苷酸TAAGTATT范围内。TAAGTATT内的突变阻断了该位点的转录起始，并导致CAT活性下降2000倍。TAAGTATT序列上游12至22个碱基的接头突变增加了在TAAGTATT内起始的RNA的稳态水平，并使CAT表达增加了50%。TAAGTATT下游以及指定非翻译RNA前导序列区域内的突变减少了TAAGTATT处的转录起始，并使CAT活性降低了2至20倍。CAT RNA的半衰期没有受到非翻译RNA前导区域突变的明显影响，因此RNA周转不是这些CAT RNA水平降低的原因。对两种突变病毒的核转录分析表明，这些突变降低了转录起始速率。因此，转录起始似乎是多角体蛋白基因调控的主要方式。这些数据确定了TAAGTATT以及指定非翻译mRNA前导序列在转录起始中的启动子相关作用。提出了一个病毒诱导的RNA聚合酶区分晚期和极晚期起始位点的模型。
