Broyles S S, Li J
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
J Virol. 1993 Sep;67(9):5677-80. doi: 10.1128/JVI.67.9.5677-5680.1993.
The vaccinia virus early transcription factor (VETF), in addition to the viral RNA polymerase, is required for efficient transcription of early genes in vitro. VETF is a heterodimeric protein that binds specifically to early gene promoters. In order to localize the VETF DNA binding domain, we have used photoreactive oligonucleotide probes with the sequence of the vaccinia virus growth factor promoter. The probes consisted of double-stranded oligonucleotides incorporating radiolabeled dAMP and 5-bromo-dUMP into sequences of the promoter known to contact VETF. Irradiation of a DNA probe having these nucleotides located upstream of the transcription start site in the presence of VETF resulted in the transfer of label to a polypeptide that comigrated with the small subunit of VETF. The label transfer reaction was shown to occur with the recombinant VETF small subunit in the absence of the large subunit. These results indicate that the small subunit comprises at least part of the VETF DNA binding domain and contacts the promoter in the region upstream of the transcription start site.
痘苗病毒早期转录因子（VETF），除病毒RNA聚合酶外，在体外是早期基因高效转录所必需的。VETF是一种异二聚体蛋白，它特异性结合早期基因启动子。为了定位VETF的DNA结合结构域，我们使用了具有痘苗病毒生长因子启动子序列的光反应性寡核苷酸探针。这些探针由双链寡核苷酸组成，将放射性标记的dAMP和5-溴-dUMP掺入已知与VETF接触的启动子序列中。在VETF存在的情况下，照射转录起始位点上游具有这些核苷酸的DNA探针，导致标记转移到与VETF小亚基共迁移的一种多肽上。在没有大亚基的情况下，标记转移反应也能与重组VETF小亚基发生。这些结果表明，小亚基至少构成VETF DNA结合结构域的一部分，并在转录起始位点上游区域与启动子接触。
