Chen M C, Cheng M C, Chen S C
Institute of Biochemistry, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China.
Plant Cell Physiol. 1993 Jun;34(4):577-84.
Transcriptional analysis of the promoter of rice psaA-psaB-rps14 operon was performed with Bal 31 deleted mutants in spinach chloroplast extracts. Two functional promoters in this operon (denoted as "-175" and "-129" promoters) were revealed to be utilized by the spinach transcription machinery; however only the "-175" promoter is utilized in vivo. The existence of psaA promoter-specific binding factors in the chloroplast extracts was indicated by competitive gel retardation assay and exonuclease III protection analysis. After ammonium sulfate fractionation of the extracts followed by heparin-agarose column chromatography, the sequence-specific binding factors were characterized to be 66-kDa and 31-kDa polypeptides. Southwestern analysis demonstrated that the two protein factors interact independently with the psaA promoter-containing DNA fragment.
利用菠菜叶绿体提取物中的Bal 31缺失突变体对水稻psaA-psaB-rps14操纵子的启动子进行了转录分析。该操纵子中的两个功能性启动子（分别记为“-175”和“-129”启动子）被发现可被菠菜转录机制利用；然而，体内仅利用“-175”启动子。竞争性凝胶阻滞试验和核酸外切酶III保护分析表明叶绿体提取物中存在psaA启动子特异性结合因子。对提取物进行硫酸铵分级分离，然后进行肝素-琼脂糖柱层析，序列特异性结合因子被鉴定为66 kDa和31 kDa的多肽。蛋白质免疫印迹分析表明这两种蛋白质因子独立地与含有psaA启动子的DNA片段相互作用。
