Harris N, Rosales R, Moss B
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2860-4. doi: 10.1073/pnas.90.7.2860.
Cytoplasmic extracts of vaccinia virus-infected HeLa cells blocked in DNA replication were capable of transcribing templates containing the minimal promoter sequences derived from three viral intermediate-stage genes (A1L, A2L, and G8R) but not promoters from early or late genes. One of three isolated components required for transcription copurified with the viral capping enzyme, a heterodimeric protein responsible for forming the 7-methyl-guanosine(5')triphospho(5')nucleoside [m7G(5')ppp(5')N-] structure at the 5' end of mRNAs, as had been reported using a template with another intermediate promoter [Vos, J. C., Sasker, M. & Stunnenberg, H. G. (1991) EMBO J. 10, 2553-2558]. Transcription factor activity was associated with partially, purified capping enzyme from infected cell extracts, homogeneous enzyme from purified virions, and recombinant viral enzyme from Escherichia coli. By transcribing truncated templates of different sizes, we determined that RNA chains of 35 nt were capped whereas those of 15 nt were not. Nevertheless, the capping enzyme was required for formation of short uncapped transcripts, indicating that capping and transcription initiation factor activities are independent functions.
在DNA复制中受阻的牛痘病毒感染的HeLa细胞的细胞质提取物能够转录含有源自三个病毒中期基因（A1L、A2L和G8R）的最小启动子序列的模板，但不能转录早期或晚期基因的启动子。转录所需的三个分离成分之一与病毒加帽酶共纯化，病毒加帽酶是一种异二聚体蛋白，负责在mRNA的5'端形成7-甲基鸟苷（5'）三磷酸（5'）核苷[m7G（5'）ppp（5'）N-]结构，正如使用另一个中间启动子的模板所报道的那样[Vos，J.C.，Sasker，M. & Stunnenberg，H.G.（1991）EMBO J. 10，2553 - 2558]。转录因子活性与来自感染细胞提取物的部分纯化的加帽酶、来自纯化病毒粒子的均一酶以及来自大肠杆菌的重组病毒酶相关。通过转录不同大小的截短模板，我们确定35个核苷酸的RNA链被加帽，而15个核苷酸的RNA链未被加帽。然而，加帽酶是形成短的未加帽转录本所必需的，这表明加帽和转录起始因子活性是独立的功能。
