Liere K, Maliga P
Waksman Institute, Rutgers, the State University of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA.
EMBO J. 1999 Jan 4;18(1):249-57. doi: 10.1093/emboj/18.1.249.
We report here the in vitro characterization of PrpoB-345, the tobacco rpoB promoter recognized by NEP, the phage-type plastid RNA polymerase. Transcription extracts were prepared from mutant tobacco plants lacking PEP, the Escherichia coli-like plastid-encoded RNA polymerase. Systematic dissection of a approximately 1 kb fragment determined that the rpoB promoter is contained in a 15-nucleotide segment (-14 to +1) upstream of the transcription initiation site (+1). Point mutations at every nucleotide reduced transcription, except at the -5 position which was neutral. Critical for rpoB promoter function was a CRT-motif (CAT or CGT) at -8 to -6 (transcription <30%), defining it as the promoter core. The core CAT sequence is also present in the maize rpoB promoter, which is faithfully recognized by tobacco extracts. Alignment of NEP promoters identified a CATA or TATA (=YATA) sequence at the rpoB core position, also present in plant mitochondrial promoters. Furthermore, NEP and the phage T7 RNA polymerase exhibit similar sensitivity to inhibitors of transcription. These data indicate that the nuclear RpoZ gene, identified by sequence conservation with mitochondrial RNA polymerases, encodes the NEP catalytic subunit.
我们在此报告PrpoB - 345的体外特性，PrpoB - 345是被NEP（噬菌体类型的质体RNA聚合酶）识别的烟草rpoB启动子。转录提取物是从缺乏PEP（类似大肠杆菌的质体编码RNA聚合酶）的突变烟草植株中制备的。对一个约1 kb片段进行系统剖析后确定，rpoB启动子位于转录起始位点（+1）上游的一个15个核苷酸的片段（-14至+1）中。除了在-5位置为中性外，每个核苷酸处的点突变都会降低转录水平。对于rpoB启动子功能至关重要的是位于-8至-6处的CRT基序（CAT或CGT）（转录水平<30%），将其定义为启动子核心。核心CAT序列也存在于玉米rpoB启动子中，能被烟草提取物准确识别。对NEP启动子进行比对后发现，在rpoB核心位置存在CATA或TATA（=YATA）序列，该序列也存在于植物线粒体启动子中。此外，NEP和噬菌体T7 RNA聚合酶对转录抑制剂表现出相似的敏感性。这些数据表明，通过与线粒体RNA聚合酶的序列保守性鉴定出的核RpoZ基因编码NEP催化亚基。
