Reach M, Babiss L E, Young C S
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
J Virol. 1990 Dec;64(12):5851-60. doi: 10.1128/JVI.64.12.5851-5860.1990.
An adenovirus major late promoter (MLP) has been constructed with a 4-bp alteration in the sequence which binds the transcription factor known as USF or MLTF. This upstream element has often been considered necessary and sufficient for maximal transcription of the MLP. A duplex oligonucleotide containing the mutant sequence was not capable of binding specific proteins in a band shift assay, nor was it capable of inhibiting such binding by the wild-type sequence. In an in vitro assay, the mutant sequence was incapable of inhibiting transcription from a duplex sequence containing the MLP, whereas the wild-type sequence could. These two pieces of evidence suggest that the sequence is functionally impaired. Surprisingly, a virus containing the mutant MLP had a normal replication phenotype. On more detailed examination however, we show that the mutant viral MLP was deficient in transcription at 9 h postinfection but that the rate of transcription was close to normal by 20 h postinfection. An inverted CAAT box located immediately upstream of the USF-binding element was not previously thought to be of importance to the functioning of the MLP. However, a single point mutation in the CAAT box, placed in the USF mutant background, had a marked effect upon transcription from the MLP. This result suggests that the MLP may exhibit functional redundancy in which either the USF-binding site or the CAAT box can serve as an upstream promoter element. Neither of the mutant viruses displayed any change in the levels of the divergent IVa2 transcription unit, suggesting that the levels of divergent transcription are not determined by competition for limiting transcription factors.
已构建了一种腺病毒主要晚期启动子（MLP），其与转录因子USF或MLTF结合的序列中有一个4个碱基对的改变。这个上游元件通常被认为对于MLP的最大转录是必要且充分的。在凝胶迁移实验中，含有突变序列的双链寡核苷酸不能结合特异性蛋白质，也不能抑制野生型序列的这种结合。在体外实验中，突变序列不能抑制来自含有MLP的双链序列的转录，而野生型序列可以。这两条证据表明该序列在功能上受损。令人惊讶的是，含有突变MLP的病毒具有正常的复制表型。然而，经过更详细的检查，我们发现突变的病毒MLP在感染后9小时转录不足，但在感染后20小时转录速率接近正常。位于USF结合元件紧上游的反向CAAT框以前被认为对MLP的功能不重要。然而，在USF突变背景下，CAAT框中的一个单点突变对MLP的转录有显著影响。这一结果表明，MLP可能表现出功能冗余，其中USF结合位点或CAAT框都可以作为上游启动子元件。两种突变病毒在不同的IVa2转录单元水平上均未显示任何变化，这表明不同转录的水平不是由对有限转录因子的竞争所决定的。
