Hay N, Aloni Y
Nucleic Acids Res. 1984 Feb 10;12(3):1401-14. doi: 10.1093/nar/12.3.1401.
Nuclei which were isolated from SV40 infected cells with a hypotonic detergent-free buffer were used to establish in vitro conditions which lead to transcription-termination at the attenuation site of SV40. This system allowed us to identify regulatory elements involved in transcription-termination by RNA polymerase B transcribing SV40. Transcription-termination at the attenuation site was found to be ionic strength dependent. Efficient termination occurred at low (100 mM NaCl) but not at high (100 mM (NH4)2 SO4 or 300 mM NaCl) ionic strength. When nuclei were prewashed with 300 mM NaCl, the efficiency of transcription-termination was low even when transcription was carried out at low ionic strength (100 mM NaCl). Efficient transcription-termination in the high salt prewashed nuclei was reconstituted by complementation with a high salt (300 mM NaCl) soluble factor extracted from nuclei of uninfected cells. In addition, the efficiency of transcription-termination was significantly reduced when ITP replaced GTP in the transcription reaction mixture. Our data indicate that a nuclear factor and RNA secondary structure are essential regulatory elements involved in transcription-termination by RNA polymerase B.
用低渗无去污剂缓冲液从感染SV40的细胞中分离出细胞核，用于建立体外条件，使其在SV40的衰减位点导致转录终止。该系统使我们能够鉴定参与由RNA聚合酶B转录SV40时转录终止的调控元件。发现在衰减位点的转录终止依赖于离子强度。在低离子强度（100 mM NaCl）下发生高效终止，但在高离子强度（100 mM硫酸铵或300 mM NaCl）下则不会。当细胞核用300 mM NaCl预洗涤时，即使在低离子强度（100 mM NaCl）下进行转录，转录终止效率也很低。通过用从未感染细胞的细胞核中提取的高盐（300 mM NaCl）可溶性因子互补，可重建高盐预洗涤细胞核中的高效转录终止。此外，当ITP替代转录反应混合物中的GTP时，转录终止效率显著降低。我们的数据表明，一种核因子和RNA二级结构是RNA聚合酶B转录终止所涉及的重要调控元件。
