Fuerst T R, Moss B
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
J Mol Biol. 1989 Mar 20;206(2):333-48. doi: 10.1016/0022-2836(89)90483-x.
We have analyzed the structure and stability of RNA synthesized by bacteriophage T7 RNA polymerase in mammalian cells. The T7 polymerase, expressed by a recombinant vaccinia virus, transcribed the Escherichia coli lacZ gene flanked by T7 promoter and terminator signals. The lacZ gene cassette was introduced into infected cells within either a transfected plasmid or a second recombinant vaccinia virus. The T7-lacZ transcripts, which had a half-life of approximately 75 minutes, represented approximately 30% of total cytoplasmic RNA after a 24 hour period. The latter estimation indicated a disparity between the levels of lacZ RNA and beta-galactosidase synthesis. Analysis of the T7 transcripts indicated that they were initiated correctly but that only 5 to 10% contained terminal cap structures, providing an explanation for the low translatability of the RNA. Since the 5' end of the T7 transcripts can form a stem-loop structure that might interfere with capping by vaccinia virus RNA guanylyltransferase, as well as ribosome binding and scanning, a similar vector lacking such sequences was constructed. In vitro experiments demonstrated that T7 RNA polymerase transcribed both templates with similar efficiency and that the RNA lacking the potential to form the stem-loop was capped more rapidly by the purified vaccinia virus enzyme. Nevertheless, when the stem-loop was removed, beta-galactosidase was not expressed in infected cells; moreover, no T7 transcripts could be detected, suggesting that the RNA was not made or more likely was degraded during or shortly after synthesis. There is previous evidence that vaccinia virus RNA guanylyltransferase is associated with the viral transcription complex, thereby allowing RNA synthesis and capping to occur concurrently. We suggest that a lack of coupling between the vaccinia viral RNA guanylyltransferase and bacteriophage T7 RNA polymerase delays capping of T7 transcripts and that, under these conditions, the 5'-terminal double-stranded stem is required to stabilize the nascent RNA against degradation. Although deletion of the 3' palindromic sequence specifying T7 transcriptional termination from the expression cassette resulted in RNA of more heterogeneous lengths, neither the apparent turnover rate nor translation of the RNAs was diminished appreciably.
我们分析了噬菌体T7 RNA聚合酶在哺乳动物细胞中合成的RNA的结构和稳定性。由重组痘苗病毒表达的T7聚合酶转录了两侧带有T7启动子和终止信号的大肠杆菌lacZ基因。lacZ基因盒通过转染的质粒或第二种重组痘苗病毒引入受感染细胞。T7-lacZ转录本的半衰期约为75分钟，24小时后约占细胞质总RNA的30%。后一估计表明lacZ RNA水平与β-半乳糖苷酶合成水平之间存在差异。对T7转录本的分析表明它们起始正确，但只有5%至10%含有末端帽结构，这为RNA的低翻译性提供了解释。由于T7转录本的5'端可形成茎环结构，这可能会干扰痘苗病毒RNA鸟苷酸转移酶的加帽作用以及核糖体结合和扫描，因此构建了一个缺乏此类序列的类似载体。体外实验表明，T7 RNA聚合酶以相似的效率转录两种模板，并且缺乏形成茎环潜力的RNA被纯化的痘苗病毒酶更快地加帽。然而，当去除茎环时，受感染细胞中未表达β-半乳糖苷酶；此外，未检测到T7转录本，这表明RNA未被合成，或者更有可能在合成期间或合成后不久被降解。以前有证据表明痘苗病毒RNA鸟苷酸转移酶与病毒转录复合物相关，从而使RNA合成和加帽同时发生。我们认为痘苗病毒RNA鸟苷酸转移酶与噬菌体T7 RNA聚合酶之间缺乏偶联会延迟T7转录本的加帽，并且在这些条件下，5'-末端双链茎对于稳定新生RNA防止降解是必需的。尽管从表达盒中删除指定T7转录终止的3'回文序列会导致RNA长度更加不均一，但RNA的表观周转率和翻译均未明显降低。
