GEL-ELECTROPHORETIC SEPARATION OF TRANSCRIPTION COMPLEXES - ASSAY FOR RNA-POLYMERASE SELECTIVITY AND A METHOD FOR PROMOTER MAPPING
GEL-ELECTROPHORETIC SEPARATION OF TRANSCRIPTION COMPLEXES - ASSAY FOR RNA-POLYMERASE SELECTIVITY AND A METHOD FOR PROMOTER MAPPING
复制标题
DOI：
10.1093/nar/7.7.1851
复制
发表时间：
1979-01-01
影响因子：
14.9
通讯作者：
GEIDUSCHEK, EP
中科院分区：
文献类型：
--
作者：
CHELM, BK;GEIDUSCHEK, EP
我们描述了一种用琼脂糖凝胶电泳法分析RNA聚合酶、DNA和新生RNA链的三元转录复合体的方法。这种复合体的RNA是P标记的，通过DNA荧光照片和放射自显影的简单比较，可以识别任何混合物中转录活性的DNA分子和限制性片段。描述了该方法的两个局限性：1)相对于其连接的裸DNA片段，聚合酶-DNA复合体在电泳过程中的延迟；2)非常大的三元复合体无法进入凝胶。该方法的潜在应用包括：转录单位(延伸)作图、从转录本中分离RNA分子、二核苷酸引物作图和优选模板构象的鉴定。
We describe a method for analyzing ternary transcription complexes, of RNA polymerase, DNA and nascent RNA,chains, by agarose gel electrophoresis. fchen the RNA of such complexes is P-labelled, a simple comparison of the DNA fluorogram with an autoradiogram identifies transcriptionally active DNA molecules and restriction fragments in any mixture. Two limitations on the method are described: 1) retardation during electrophoresis of polymerase-DNA complexes relative to their conjugate bare DNA fragments; 2) failure of very large ternary complexes to enter gels.The following potential applications of the method are surveyed: transcription unit (elongation) mapping, separation of RNA molecules in a mixture of transcripts, dinucleotide primer mapping and identification of preferred template conformations.