Lu H, Reach M D, Minaya E, Young C S
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
J Virol. 1997 Jan;71(1):102-9. doi: 10.1128/JVI.71.1.102-109.1997.
Previous results showed that the structure and function of the adenovirus major late promoter (MLP) can be analyzed genetically in its correct location, despite its essential role in the viral life cycle. This genetic approach was extended to investigate the in vivo role of the initiator (INR), a transcriptional element that surrounds the start site of transcription. The analysis was designed to investigate if the INR is an alternative basal element to the canonical TATA box of the MLP, its relative importance in the functioning of the promoter, and if its function was affected by upstream activating elements. Accordingly, two different mutations in the INR were created and tested in the genome, either by themselves or together with mutations in the TATA box or one of the two upstream activating elements, the upstream promoter element (UPE) and the inverted CAAT box. The mutant viruses were examined first in one-step growth experiments, and then levels of late mRNA accumulation were measured by primer extension, transcription initiation was assayed in isolated nuclei, and viral DNA accumulation was determined by Southern hybridization. Neither mutation in the INR alone had any discernible phenotypic effects but when coupled to a phenotypically silent mutation in the TATA box gave rise to viruses with growth defects that were attributable to a significantly lowered rate of transcription initiation from the MLP. These results suggest that the INR plays a role in vivo and can act as an alternative basal element in the absence of a functioning TATA box. A virus with mutations in both the INR and the UPE, although viable, likewise had a severe deficiency in transcription, suggesting that the function of the INR is affected by that of the UPE. This contrasts with the previous report that a TATA box-UPE double mutation is not recoverable in virus. In addition, the virus with mutations in both the INR and the inverted CAAT box was phenotypically wild type, unlike the previously described TATA box-CAAT box double mutant, which had a severe transcription deficiency. Taken together, the present and previous genetic results can be interpreted as evidence that in the MLP, the TATA box and the UPE are the more important of the two basal and activating elements, respectively, but that the INR and CAAT can function in transcription initiation. We consider the role of the INR in the formation of the preinitiation complex and speculate on possible protein-protein interactions.
先前的结果表明，尽管腺病毒主要晚期启动子（MLP）在病毒生命周期中起着至关重要的作用，但仍可在其正确位置对其结构和功能进行遗传学分析。这种遗传学方法被扩展用于研究起始子（INR）的体内作用，起始子是一种围绕转录起始位点的转录元件。该分析旨在研究INR是否是MLP经典TATA框的替代基础元件，其在启动子功能中的相对重要性，以及其功能是否受上游激活元件的影响。因此，在基因组中创建了两种不同的INR突变，并单独或与TATA框或两个上游激活元件之一（上游启动子元件（UPE）和反向CAAT框）中的突变一起进行测试。首先在一步生长实验中检查突变病毒，然后通过引物延伸测量晚期mRNA积累水平，在分离的细胞核中测定转录起始，并通过Southern杂交确定病毒DNA积累。单独的INR突变均未产生任何可辨别的表型效应，但当与TATA框中的表型沉默突变结合时，会产生具有生长缺陷的病毒，这归因于MLP转录起始速率显著降低。这些结果表明，INR在体内发挥作用，并且在没有功能性TATA框的情况下可以作为替代基础元件。一种INR和UPE均有突变的病毒虽然能够存活，但同样存在严重的转录缺陷，这表明INR的功能受UPE的影响。这与先前的报告形成对比，先前报告称TATA框-UPE双突变在病毒中无法恢复。此外，INR和反向CAAT框均有突变的病毒在表型上是野生型，这与先前描述的TATA框-CAAT框双突变体不同，后者存在严重的转录缺陷。综上所述，目前和先前的遗传学结果可以解释为证据，表明在MLP中，TATA框和UPE分别是两个基础元件和激活元件中更重要的元件，但INR和CAAT可以在转录起始中发挥作用。我们考虑了INR在起始前复合物形成中的作用，并推测了可能的蛋白质-蛋白质相互作用。
