嗜盐古菌bop基因启动子中TATA盒和上游激活序列的饱和诱变
Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter.
作者信息
Baliga N S, DasSarma S
机构信息
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
出版信息
J Bacteriol. 1999 Apr;181(8):2513-8. doi: 10.1128/JB.181.8.2513-2518.1999.
Abstract
Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon Halobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum- bop) Halobacterium mutant for purple (Pum+ bop+) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5'-tyT(T/a)Ta-3', corresponding to the promoter TATA box element 30 to 25 bp 5' of the transcription start site. A putative UAS, 5'-ACCcnactagTTnG-3', located 52 to 39 bp 5' of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.
摘要
简并寡核苷酸用于在三个7碱基片段中随机化53碱基最小bop启动子的21个碱基，包括假定的TATA盒和上游激活序列（UAS）。将诱变的bop启动子、野生型结构基因和转录终止子插入到能够在嗜盐古菌盐生盐杆菌S9菌株中复制的穿梭质粒中。通过在琼脂平板上筛选橙色（Pum - bop）盐生盐杆菌突变体的转化子以获得紫色（Pum + bop +）菌落来分离活性启动子，并分析bop mRNA和/或细菌视紫红质含量。序列分析产生了共有序列5'-tyT(T/a)Ta-3'，对应于转录起始位点5'端30至25碱基处的启动子TATA盒元件。在转录起始位点5'端52至39碱基处发现的一个假定的UAS，即5'-ACCcnactagTTnG-3'，在活性启动子中是保守的。本研究为TATA盒和UAS对bop启动子活性的需求提供了直接证据。
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