Lang D, Stamminger T
Institut für Klinische und Molekulare Virologie der Universität Erlangen-Nürnberg, Germany.
Nucleic Acids Res. 1994 Aug 25;22(16):3331-8. doi: 10.1093/nar/22.16.3331.
The 86 kDa immediate early-2 protein (IE2, IE86) of human cytomegalovirus (HCMV) is a multifunctional polypeptide that can regulate gene expression both positively and negatively. In particular, it represses its own mRNA synthesis by binding directly to a sequence element, termed cis repression signal (CRS), that is located between the TATA box and the transcriptional start site of the major IE enhancer/promoter of HCMV. Here, we provide evidence that IE86, unlike most sequence-specific DNA-binding proteins, interacts primarily within the minor groove of the DNA helix. This was shown by hydroxyl radical and methylation interference assays. In addition, binding studies with inosine-substituted oligonucleotides which have an altered major groove morphology without changing the surface of the minor groove, confirmed the results obtained in interference analyses. This establishes IE86 as a member of a small group of DNA binding proteins that interact with A - T rich sequences within the minor groove and which also includes the TATA-box binding protein TBP. Remarkably, IE86 and TBP are able to bind simultaneously in an immediate vicinity at the major IE enhancer/promoter of HCMV. As minor groove binding proteins are known to bend DNA heavily this could contribute to the observed negative regulation of transcription by IE86.
人巨细胞病毒（HCMV）的86 kDa即刻早期2蛋白（IE2，IE86）是一种多功能多肽，可对基因表达进行正向和负向调控。特别地，它通过直接结合位于HCMV主要IE增强子/启动子的TATA盒和转录起始位点之间的一个序列元件（称为顺式抑制信号，CRS）来抑制自身mRNA的合成。在此，我们提供证据表明，与大多数序列特异性DNA结合蛋白不同，IE86主要在DNA螺旋的小沟内相互作用。这通过羟自由基和甲基化干扰试验得以证明。此外，与肌苷取代的寡核苷酸进行的结合研究证实了干扰分析的结果，这些寡核苷酸的大沟形态发生了改变，但小沟表面未变。这确立了IE86作为一小类DNA结合蛋白的成员，这类蛋白与小沟内富含A - T的序列相互作用，其中还包括TATA盒结合蛋白TBP。值得注意的是，IE86和TBP能够在HCMV主要IE增强子/启动子的紧邻位置同时结合。由于已知小沟结合蛋白会使DNA发生严重弯曲，这可能导致观察到的IE86对转录的负调控。
