Efficient transcription of the EBER2 gene depends on the structural integrity of the RNA
Efficient transcription of the EBER2 gene depends on the structural integrity of the RNA
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DOI：
10.1261/rna.2176603
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发表时间：
2003-04-01
期刊：
影响因子：
4.5
通讯作者：
Benecke, BJ
中科院分区：
文献类型：
--
作者：
Dümpelmann, E;Mittendorf, H;Benecke, BJ
一个3 ′-截短的EBER 2 RNA基因，虽然含有所有以前鉴定的启动子元件，但当与异源终止子序列融合时，在体外和体内显示出显著降低的转录速率。还观察到影响EBER 2基因5 '或3'末端序列的双点突变的失活。然而，这些突变体的野生型活性可以通过EBER 2 RNA序列的相反链的补偿突变来恢复。如果异源终止子适于与构建体的起始元件互补，则用3 ′-截短的EBER 2基因实现类似的拯救。然而，RNA末端的双链性不足以使这些基因构建体具有高转录活性。相反，使用不可折叠的间隔区，分离5 '-和3'-茎环结构，证明EBER 2 RNA末端的空间接近是必需的。此外，体外合成的野生型和突变体RNA的衰减动力学表明，观察到的效果不能用改变的转录稳定性来解释。最后，单轮转录证实了突变基因表达的减少不是由初级起始反应的减少引起的。此外，不同的肌氨酸浓度表明，再起始的速率明显受到突变EBER 2基因的影响。总之，这些结果表明，这种病毒RNA的二级结构是宿主细胞RNA聚合酶III有效转录EBER 2基因的主要决定因素。
A 3'-truncated EBER2 RNA gene, although containing all previously identified promoter elements, revealed drastically reduced transcription rates in vitro and in vivo when fused to a heterologous terminator sequence. Inactivations were also observed with double point mutations affecting 5'- or 3'-end sequences of the EBER2 gene. However, wild-type activity of these mutants could be restored by compensatory mutations of the opposite strand of the EBER2 RNA sequence. A similar rescue was achieved with the 3'-truncated EBER2 gene, if the heterologous terminator was adapted for complementarity to the initiator element of the construct. Yet, double-strandedness alone of the RNA ends was not sufficient for high transcriptional activity of these gene constructs. Rather, the use of a nonrefoldable spacer, separating the 5'- and 3'-stem-loop structures, demonstrated that spatial proximity of the ends of EBER2 RNA was required. Furthermore, decay kinetics of wild-type and mutant RNA synthesized in vitro indicated that the effects observed could not be explained by altered transcript stability. Finally, single-round transcription confirmed that the reduced expression of mutant genes was not caused by decreased primary initiation reactions. In addition, differential sarcosyl concentrations demonstrated that the rate of reinitiation clearly was affected with the mutant EBER2 genes. Together, these results indicate that the secondary structure of this viral RNA represents a major determinant for efficient transcription of the EBER2 gene by host cell RNA polymerase III.