Kestermann M, Neukirchen S, Kloppstech K, Link G
Plant Cell Physiology and Molecular Biology, University of Bochum, D-44780 Bochum, Germany.
Nucleic Acids Res. 1998 Jun 1;26(11):2747-53. doi: 10.1093/nar/26.11.2747.
Plant chloroplasts contain transcription factors that functionally resemble bacterial sigma factors. We have cloned the full-length cDNA from mustard (Sinapis alba) for a 53 kDa derived polypeptide that contains similarity to regions 1.2-4.2 of sigma70-type factors. The amino acid sequence at the N-terminus has characteristics of a chloroplast transit peptide. An in vitro synthesized polypeptide containing this region was shown to be imported into the chloroplast and processed. The recombinant factor lacking the N-terminal extension was expressed in Escherichia coli and purified. It confers the ability on E.coli core RNA polymerase to bind specifically to a DNA fragment that contains the chloroplast psbA promoter. Transcription of the psbA template by E.coli core enzyme in the presence of recombinant SIG1 results in enhanced formation of transcripts of the size expected for correct initiation at the in vivo start site. Together, these data suggest that the mature protein acts as one of the chloroplast transcription factors in mustard. RNA gel blot hybridization reveals a transcript at approximately 1.8 kb, which is more abundant in light-grown than in dark-grown mustard seedlings.
植物叶绿体含有在功能上类似于细菌σ因子的转录因子。我们已经从芥菜（白芥）中克隆了一个53 kDa衍生多肽的全长cDNA，该多肽与σ70型因子的1.2 - 4.2区域具有相似性。N端的氨基酸序列具有叶绿体转运肽的特征。含有该区域的体外合成多肽被证明可导入叶绿体并进行加工。缺失N端延伸的重组因子在大肠杆菌中表达并纯化。它赋予大肠杆菌核心RNA聚合酶特异性结合包含叶绿体psbA启动子的DNA片段的能力。在重组SIG1存在下，大肠杆菌核心酶对psbA模板的转录导致在体内起始位点正确起始预期大小的转录本形成增加。这些数据共同表明，成熟蛋白作为芥菜叶绿体转录因子之一发挥作用。RNA凝胶印迹杂交显示约1.8 kb处有一个转录本，在光下生长的芥菜幼苗中比在黑暗中生长的更丰富。
