Watson J B, Chandler D W, Gralla J D
Nucleic Acids Res. 1984 Jul 11;12(13):5369-84. doi: 10.1093/nar/12.13.5369.
In vitro transcription of cleaved SV40 DNA with calf thymus RNA polymerase reveals a discrete transcript. The pattern of resistance to the inhibitor alpha-amanitin identifies the RNA as a product of RNA polymerase III transcription. The RNA is shown to initiate artificially near a DNA terminus created by cleavage and to terminate specifically near a cluster of 8 thymidine residues within the SV40 control region. Faithfully initiated transcripts cannot be detected using the calf thymus enzyme, supporting the idea that polymerase III termination can be accomplished by an initiation-deficient enzyme. Transcription of SV40 DNA in a HeLa cell lysate also leads to specific polymerase III transcription. When PvuII-cleaved DNA is the template, the same RNA is produced as with the calf thymus enzyme. At the lowered lysate concentration known to activate certain AluI-family transcripts, a collection of SV40 polymerase III transcripts is also produced. These do not depend on restriction cleavage of the DNA and thus arise from transcription of intact DNA.
用小牛胸腺RNA聚合酶对切割后的SV40 DNA进行体外转录，可得到一条离散的转录本。对抑制剂α-鹅膏蕈碱的抗性模式表明该RNA是RNA聚合酶III转录的产物。研究表明，该RNA在切割产生的DNA末端附近人工起始，并在SV40控制区内的8个胸腺嘧啶残基簇附近特异性终止。使用小牛胸腺酶无法检测到忠实起始的转录本，这支持了聚合酶III终止可由起始缺陷型酶完成的观点。在HeLa细胞裂解物中对SV40 DNA进行转录也会导致特异性的聚合酶III转录。当用PvuII切割的DNA作为模板时，产生的RNA与小牛胸腺酶产生的相同。在已知可激活某些AluI家族转录本的较低裂解物浓度下，也会产生一系列SV40聚合酶III转录本。这些转录本不依赖于DNA的限制性切割，因此来自完整DNA的转录。
