转录起始因子sigma70在转录延伸过程中的保留：单分子分析
Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis.
作者信息
Kapanidis Achillefs N, Margeat Emmanuel, Laurence Ted A, Doose Sören, Ho Sam On, Mukhopadhyay Jayanta, Kortkhonjia Ekaterine, Mekler Vladimir, Ebright Richard H, Weiss Shimon
机构信息
Department of Chemistry and Biochemistry, and California Nanosystems Institute, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095, USA.
出版信息
Mol Cell. 2005 Nov 11;20(3):347-56. doi: 10.1016/j.molcel.2005.10.012.
We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and sigma70 contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, approximately 70%-90%, of early elongation complexes retain sigma70 and that a determinant for sigma70 recognition in the initial transcribed region increases sigma70 retention in early elongation complexes. The results establish that a significant fraction, approximately 50%-60%, of mature elongation complexes retain sigma70 and that a determinant for sigma70 recognition in the initial transcribed region does not appreciably affect sigma70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma70, the half-life of sigma70 retention is long relative to the time-scale of elongation, suggesting that some complexes may retain sigma70 throughout elongation.
我们报告了一种单分子检测方法，该方法可同时确定蛋白质复合物相对于DNA的转位位置以及复合物的亚基化学计量。我们应用该检测方法在体外确定细菌转录延伸复合物的转位位置和σ70含量。结果证实了整体结果，表明很大一部分（约70%-90%）的早期延伸复合物保留了σ70，并且初始转录区域中σ70识别的决定因素增加了早期延伸复合物中σ70的保留率。结果表明，相当一部分（约50%-60%）的成熟延伸复合物保留了σ70，并且初始转录区域中σ70识别的决定因素对成熟延伸复合物中σ70的保留没有明显影响。结果进一步表明，在保留σ70的成熟延伸复合物中，σ70保留的半衰期相对于延伸的时间尺度较长，这表明一些复合物可能在整个延伸过程中都保留σ70。
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