Roberge M, Dahmus M E, Bradbury E M
Department of Biological Chemistry School of Medicine, University of California, Davis 95616.
J Mol Biol. 1988 Jun 5;201(3):545-55. doi: 10.1016/0022-2836(88)90636-5.
The relative distribution of transcriptionally active and inactive RNA polymerases I and II between the nuclear matrix/scaffold and chromosomal loops of HeLa cells was determined. Total RNA polymerase was assessed by immunoblotting and transcribing RNA polymerase by a photoaffinity labeling technique in isolated nuclei. Nuclear matrix/scaffold was isolated by three methods using high-salt, intermediate-salt or low-salt extraction. The distribution of RNA polymerases I and II were very similar within each of the methods, but considerable differences in distributions were found between the different preparation methods. Either intermediate-salt or high-salt treatment of DNase I-digested nuclei showed significant association of RNA polymerases with the nuclear matrix. However, intermediate-salt followed by high-salt treatment released all transcribing and non-transcribing RNA polymerases. Nuclear scaffolds isolated with lithium diiodosalicylate (low-salt) contained very little of the RNA polymerases. This treatment, however, caused the dissociation of RNA polymerase II transcription complexes. These results show unambiguously that RNA polymerases, both in their active and inactive forms, are not nuclear matrix proteins. The data support models in which the transcriptional machinery moves around DNA loops during transcription.
测定了HeLa细胞核基质/支架与染色体环中转录活性和非活性的RNA聚合酶I和II的相对分布。通过免疫印迹法评估总RNA聚合酶，并在分离的细胞核中使用光亲和标记技术评估转录中的RNA聚合酶。使用高盐、中盐或低盐提取的三种方法分离核基质/支架。在每种方法中，RNA聚合酶I和II的分布非常相似，但在不同的制备方法之间发现了分布上的显著差异。对DNase I消化的细胞核进行中盐或高盐处理均显示RNA聚合酶与核基质有显著关联。然而，先进行中盐处理再进行高盐处理会释放所有转录和非转录的RNA聚合酶。用二碘水杨酸锂（低盐）分离的核支架中RNA聚合酶含量极少。然而，这种处理导致RNA聚合酶II转录复合物解离。这些结果明确表明，无论是活性形式还是非活性形式的RNA聚合酶都不是核基质蛋白。这些数据支持了转录过程中转录机制围绕DNA环移动的模型。
