Using the 3' end of the L1 mRNA family of adenovirus 2 (Ad2) as a model system, we investigated transcription elongation following a poly(A) signal in a cell-free system. The results show that RNA polymerase II can halt transcription elongation at a T-rich stretch in the non-coding DNA strand 20 nucleotides downstream of the poly(A) signal. The block to transcription elongation is enhanced when Sarkosyl is included in the elongation reaction. Deletion studies narrowed the region which directs the elongation block at the T-rich stretch, to an upstream fragment of 53 nucleotides that is very dA-rich and also contains a functional poly(A) signal. The deletion studies and analysis by site-directed mutagenesis indicate that in the present system, RNA secondary structure, the stretch of T's and the poly(A) signal are not the dominant elements responsible for the elongation block. The block to transcription elongation at the T-rich stretch was also shown to be 5 times more effective in an uninfected extract than in an Ad2 infected extract, which is reminiscent of the in vivo situation and is consistent with the suggestion that a trans-acting factor is involved in modulating the elongation block at the T-rich stretch.

译文

使用腺病毒2 (Ad2) 的L1 mRNA家族的3' 末端作为模型系统,我们研究了无细胞系统中poly (a) 信号后的转录伸长。结果表明,RNA聚合酶II可以在poly (a) 信号下游的非编码DNA链20个核苷酸中终止富含T的转录延伸。当伸长率反应中包括Sarkosyl时,对转录延伸的阻滞会增强。缺失研究将引导富含T的延伸区缩小到53个核苷酸的上游片段,该片段非常富含dA,并且还包含功能性poly (a) 信号。通过定点诱变进行的缺失研究和分析表明,在本系统中,RNA二级结构,T的延伸和poly(A) 信号不是导致伸长阻滞的主要因素。还显示,在未感染的提取物中,在富含T的拉伸处对转录延伸的阻滞比在Ad2感染的提取物中有效5倍,这让人想起体内的情况,并且与反式作用因子参与调节富含T的拉伸处的伸长阻滞的建议一致。

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