Role of RNA polymerase subunit RPB 5 in transcriptional complex formation and Dranscriptional control

我们以前的发现，HBx直接与RPB 5，RNA聚合酶的共同亚基，相互作用，这意味着HBx直接调节RNA聚合酶的功能。本项目研究了HBx对RPB 5转录调控的分子机制。1.在体外和体内实验中，我们发现HBx和RPB 5都能与TFIIB特异性结合。我们可以在体内和体外检测由RPB 5、HBx和TFIIB组成的三元复合物。2.一些在反式作用活性方面严重受损的HBx取代突变体以相互排斥的方式表现出与TFIIB或RPB 5的结合亲和力降低，提示HBx的反式激活需要RPB 5和TFIIB的相互作用。结果表明HBx是一种新的病毒调节剂，通过与RPB 5和TFIIB的通讯稳定RNA聚合酶和TFIIB之间的结合，促进转录起始。TFIIB.3.我们研究了当HBx被募集到远端顺式元件时是否作为转录激活因子，以及HBx是否对体外转录产生积极影响。结果表明，HBx不能作为一个转录的反式激活因子，但可以作为一个辅助因子参与转录通过蛋白质-蛋白质相互作用。4.为了进一步了解RPB 5的功能，我们尝试通过far-Western blotting直接克隆RPB 5结合蛋白。我们成功地分离出了一种未报道的独特的cCNA。该基因命名为RPB 5介导蛋白（RMP）。初步实验表明，RMP可干扰哺乳动物细胞中HBx的反式激活。结果提示，RMP对HBx有拮抗作用。正在进一步表征RMP。

Our previous finding that HBx directly interacts with RPB5, a common subunit of RNA polymerases, implies that HBx modulates the function of RNA polymerase directly. This project addressed the molecular mechanism of transcriptional modulation of RPB5 by HBx. 1.We found that both HBx and RPB5 specifically bound to TFIIB in vitro and in vivo. We could detect the ternary complex consisting of RPB5, HBx, and TFIIB in vivo and in vitro. 2.Some HBx substitution mutants, which were severely impaired in transacting activity, exhibited reduced binding affinity with either TFIIB or RPB5 in a mutually exclusive manner, suggesting that HBx transactivation requires the interactions of both RPB5 and TFIIB.The results indicate that HBx is a novel virus modulator that facilitates transcriptional initiation by stabilizing the association between RNA polymerase and TFIIB through communication with RPB5 and TFIIB.3.We addressed whether HBx acts as a transcriptional activator when it is recruited to a distal cis-element, and whether HBx positively affects in vitro transcription. The results indicated that HBx can not act as a transcriptional transactivator but can act as a cofactor involving in transcription through protein-protein interaction. 4.To understand the function of RPB5, we tried to isolate RPB5-binding protein by direct cDNA cloning using far-Western blotting. We successed to isolate a cCNA redundantly which was found unreported and unique. The gene was designated as RPB5 mediating protein (RMP). Preliminarily experiments showed that RMP may interfer transactivation of HBx in mammalian cells. The results suggest that RMP may antagonize HBx. Further characterization of RMP is on going.