Imamura Sousuke, Asayama Munehiko, Shirai Makoto
Laboratory of Molecular Genetics, College of Agriculture, Ibaraki University, Ami, Inashiki, Ibaraki 300-0393, Japan.
Genes Cells. 2004 Dec;9(12):1175-87. doi: 10.1111/j.1365-2443.2004.00808.x.
The RNA polymerase (RNAP) core enzyme of cyanobacterium Synechocystis sp. strain PCC 6803 was reconstituted with overproduced recombinant subunits and purified with C-terminal histidine-tagged RpoA. The core enzyme with purified a sigma factor, SigA/SigD or SigB, allowed specific in vitro transcription from the light-inducible psbA2 or the dark-/heat-inducible lrtA/hspA promoters, respectively. Further analysis using a mutant psbA2 promoter revealed that the -35 hexamer of the promoter was essential for SigA but not SigD. Similar but distinct patterns of psbA2 transcription were found for two types of RNAP, cyanobacterial (alpha2betabeta'gamma) and E. coli (alpha2betabeta') core enzymes. Specific binding of PCC 6803 RpoC2 (beta') to E. coli core enzyme and its contribution to efficient psbA2 transcription by RNAP-SigA/D suggest that this subunit could confer an important role on the cyanobactrial RNAP. Differences in affinity and specificity among cyanobacterial sigma factors for the core enzyme and promoters were discussed.
用过量表达的重组亚基重建了集胞藻PCC 6803的RNA聚合酶（RNAP）核心酶，并用C端带组氨酸标签的RpoA进行纯化。用纯化的σ因子SigA/SigD或SigB重建的核心酶分别允许从光诱导型psbA2或暗/热诱导型lrtA/hspA启动子进行特异性体外转录。使用突变的psbA2启动子进行的进一步分析表明，启动子的-35六聚体对SigA是必需的，但对SigD不是必需的。对于两种类型的RNAP，即蓝藻（α2ββ′γ）和大肠杆菌（α2ββ′）核心酶，发现了相似但不同的psbA2转录模式。PCC 6803 RpoC2（β′）与大肠杆菌核心酶的特异性结合及其对RNAP-SigA/D高效转录psbA2的贡献表明，该亚基可能赋予蓝藻RNAP重要作用。讨论了蓝藻σ因子对核心酶和启动子的亲和力和特异性差异。
