Ziff E B, Evans R M
Cell. 1978 Dec;15(4):1463-75. doi: 10.1016/0092-8674(78)90070-3.
During the late stage of adenovirus 2 infection, RNA chains are initiated at a site near coordinate 16 (Evans et al., 1977) and transcribed approximately 30,000 nucleotides to the far end of the genome at coordinate 100. Late mRNAs processed from these transcripts contain a common spliced tripartite leader (Berget, Moore and Sharp, 1977; Chow et al., 1977a) encoded at approximately 16, 20 and 27, and protein coding sequences which map downstream. This report maps the late promoter and the capped 5' end of nuclear and cytoplasmic RNAs from this transciption unit, and analyzes their structures. We show that nascent RNA chains pulse-labeled in vivo are initiated at coordinate 16.5 +/- 0.5 and contain the sequences intervening between the leader segments. We map the capped 5' terminus of late nuclear transcripts at a site between 16.4 and 16.6 by aligning T1 RNAase oligonucleotides from nuclear RNA with the DNA sequence of the promoter region. The structure of the first eleven residues of the capped 5' terminus of late mRNA was determined by direct RNA sequencing. This structure corresponds exactly to a DNA sequence at coordinate 16.4 and precisely positions the mRNA cap template within the promoter region. These results suggest that the promoter and the cap template sites are coincident, and that the initiating residues of the primary transcript are precursors of the capped 5' end of mRNA. Residues removed from transcripts by splicing were identified. These plus caps were detected in large polyadenylated nuclear RNA, indicating that capping and polyadenylation can occur on unspliced molecules. Residues retained in the mRNA first leader contain a nine residue sequence adjacent to the cap which is complementary to the 3' end of 18S rRNA, suggesting that the first leader functions in ribosome binding. Nucleotide sequences from the promoter region are compared with cellular counterparts. Strong homologies at cap sites and splice points suggest that for the noted cases, the virus and cell share closely related mechanisms for mRNA 5' end synthesis and splicing.
在腺病毒2感染后期，RNA链在坐标16附近的位点起始（埃文斯等人，1977年），并转录约30,000个核苷酸至基因组坐标100处的远端。从这些转录本加工而来的晚期mRNA含有一个共同的剪接三联体前导序列（伯杰、穆尔和夏普，1977年；周等人，1977a），其编码位置约在坐标16、20和27处，以及位于下游的蛋白质编码序列。本报告绘制了该转录单元的晚期启动子以及核RNA和细胞质RNA的加帽5'端图谱，并分析了它们的结构。我们发现，体内脉冲标记的新生RNA链在坐标16.5±0.5处起始，并包含前导序列之间的间隔序列。通过将来自核RNA的T1核糖核酸酶寡核苷酸与启动子区域的DNA序列比对，我们将晚期核转录本的加帽5'末端定位在16.4和16.6之间的一个位点。通过直接RNA测序确定了晚期mRNA加帽5'末端前11个残基的结构。该结构与坐标16.4处的DNA序列完全对应，并精确地将mRNA帽模板定位在启动子区域内。这些结果表明，启动子和帽模板位点重合，并且初级转录本的起始残基是mRNA加帽5'端的前体。鉴定了通过剪接从转录本中去除的残基。这些加帽结构在大量多聚腺苷酸化的核RNA中被检测到，表明加帽和多聚腺苷酸化可以在未剪接的分子上发生。保留在mRNA第一个前导序列中的残基包含一个与帽相邻的九个残基序列，该序列与18S rRNA的3'端互补，表明第一个前导序列在核糖体结合中起作用。将启动子区域的核苷酸序列与细胞中的对应序列进行了比较。帽位点和剪接位点的高度同源性表明，对于所提及的情况，病毒和细胞在mRNA 5'端合成和剪接方面具有密切相关的机制。
