Concino M F, Lee R F, Merryweather J P, Weinmann R
Nucleic Acids Res. 1984 Oct 11;12(19):7423-33. doi: 10.1093/nar/12.19.7423.
Mutagenized DNA templates and HeLa whole cell extracts were used to study the effects of promoter-specific base changes on in vitro transcription. DNA templates where the initiating adenine (+1) was changed to thymidine (AT+1) in the adenovirus 2 major late transcription unit were transcribed with 50% efficiency of the unaltered template. We have described a mutant at the TATA box, where the A at position -28 was changed to a C (AC-28). Transcription efficiency was reduced to less than 20% of control in the AC-28 mutant (Concino et al., 1983, J. Biol. Chem. 258: 8493-8496). Primer extension analysis revealed increased 5' end heterogeneity for in vitro transcripts derived from AC-28 and AT+1 DNA templates. Specific transcription was completely abolished from AT+1 DNA templates when a second change was introduced within the TATA sequence, creating a double mutant (AC-28 . AT+1). Neither the AC-28 nor the AT+1 change alone had such an effect, suggesting a coordinated interaction in transcription initiation involving both the TATA box and the initiation site.
利用诱变的DNA模板和HeLa全细胞提取物研究启动子特异性碱基变化对体外转录的影响。在腺病毒2主要晚期转录单元中，起始腺嘌呤（+1）被改变为胸腺嘧啶（AT+1）的DNA模板，其转录效率为未改变模板的50%。我们描述了一个位于TATA框的突变体，其中-28位的A被改变为C（AC-28）。在AC-28突变体中，转录效率降至对照的20%以下（Concino等人，1983年，《生物化学杂志》258:8493-8496）。引物延伸分析显示，源自AC-28和AT+1 DNA模板的体外转录本的5'端异质性增加。当在TATA序列内引入第二个变化，创建一个双突变体（AC-28.AT+1）时，AT+1 DNA模板的特异性转录完全被消除。单独的AC-28或AT+1变化都没有这种效果，这表明在转录起始过程中，TATA框和起始位点之间存在协同相互作用。
