STRUCTURE AND STABILITY OF MESSENGER-RNA SYNTHESIZED BY VACCINIA VIRUS-ENCODED BACTERIOPHAGE T7 RNA-POLYMERASE IN MAMMALIAN-CELLS - IMPORTANCE OF THE 5' UNTRANSLATED LEADER
STRUCTURE AND STABILITY OF MESSENGER-RNA SYNTHESIZED BY VACCINIA VIRUS-ENCODED BACTERIOPHAGE T7 RNA-POLYMERASE IN MAMMALIAN-CELLS - IMPORTANCE OF THE 5' UNTRANSLATED LEADER
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DOI：
10.1016/0022-2836(89)90483-x
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发表时间：
1989-03-20
影响因子：
5.6
通讯作者：
MOSS, B
中科院分区：
文献类型：
--
作者：
FUERST, TR;MOSS, B
我们分析了哺乳动物细胞中由噬菌体T7 RNA聚合酶合成的RNA的结构和稳定性。由重组牛痘病毒表达的T7聚合酶转录两侧为T7启动子和终止子信号的大肠杆菌lacZ基因。将lacZ基因盒引入转染质粒或第二重组牛痘病毒内的感染细胞中。半衰期约为75分钟的T7-lacZ转录物在24小时后占总细胞质RNA的约30%。后一种估计表明lacZ RNA和β-lacZ RNA水平之间的差异。半乳糖苷酶合成对T7转录本的分析表明，它们被正确启动，但只有5至10%包含端帽结构，这为RNA的低翻译性提供了解释。由于T7转录物的5 "末端可以形成茎环结构，其可能干扰牛痘病毒RNA鸟苷酰转移酶的加帽以及核糖体结合和扫描，因此构建了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，并且缺乏形成茎环的潜力的RNA被纯化的牛痘病毒酶更快地加帽。然而，当茎环被移除时，β-半乳糖苷酶在感染的细胞中不表达;此外，未检测到T7转录本，表明RNA在细胞中不表达;此外，未检测到T7转录本，表明RNA在合成期间或合成后不久未产生或更可能降解。有宝贵的证据表明，牛痘病毒RNA鸟苷酰转移酶与病毒转录复合物相关，从而允许RNA合成和加帽同时发生。我们认为牛痘病毒RNA鸟苷酰转移酶和噬菌体T7 RNA聚合酶之间缺乏偶联延迟了T7转录物的加帽，并且在这些条件下，需要5 "-末端双链茎来稳定新生RNA以防降解。尽管从表达盒中删除3 "回文序列T7转录终止导致RNA的长度更不均匀，但RNA的表观周转率和翻译都没有明显减少。
We have analyzed the structure and stability of RNA synthesized by cacteriophage 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 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 precious evidence that vaccinia virus RNA guanylyl- transferase 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 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.