Gu X, Marzluff W F
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, 27599, USA.
Nucleic Acids Res. 1996 Oct 1;24(19):3797-805. doi: 10.1093/nar/24.19.3797.
The highly expressed mouse histone H2a-614 gene is located 800 nt 5' of the histone H3-614 gene. There is a 140 nt sequence located 500 nt from the end of the H2-614 mRNA which has been defined as a transcription termination site for RNA polymerase II. We established an in vitro transcription system in which both 3' end processing and transcription termination occur. A template containing the adenovirus major late promoter, a portion of the histone H2a-614 coding region, its 3' processing signal, followed by the transcription termination site was transcribed in a nuclear extract prepared from mouse myeloma cells. Some of the transcripts synthesized in the extract were cleaved at the histone processing site in a reaction which was dependent both on the hairpin binding factor and the U7 snRNP. The efficiency of histone 3' end formation was similar both on synthetic transcripts and transcripts synthesized by RNA polymerase II. Defined transcripts, which were not processed and which mapped to the transcription termination site, were released from the template, suggesting that they were formed by transcription termination. Termination in vitro was dependent on a functional histone processing signal.
高表达的小鼠组蛋白H2a - 614基因位于组蛋白H3 - 614基因5'端800个核苷酸处。在距离H2 - 614 mRNA末端500个核苷酸处有一个140个核苷酸的序列，该序列已被定义为RNA聚合酶II的转录终止位点。我们建立了一个体外转录系统，其中3'端加工和转录终止都能发生。一个包含腺病毒主要晚期启动子、组蛋白H2a - 614编码区的一部分、其3'加工信号，随后是转录终止位点的模板，在从小鼠骨髓瘤细胞制备的核提取物中进行转录。提取物中合成的一些转录本在组蛋白加工位点被切割，该反应既依赖于发夹结合因子，也依赖于U7 snRNP。组蛋白3'端形成的效率在合成转录本和由RNA聚合酶II合成的转录本上相似。未加工且定位到转录终止位点的特定转录本从模板上释放，表明它们是由转录终止形成的。体外终止依赖于功能性的组蛋白加工信号。
