Role of RNA polymerase, II submit 5 (RPB5) in activated transcription, and function of a novel transcriptional cofactor, RMP.

位于pol II下颚尖端的RPB5参与激活转录，并与基础转录因子和乙型肝炎病毒X蛋白（HBx）相互作用。pol II晶体模型预测RPB5靠近转录起始位点下游的DNA。1) RPB5与RAP30和RAP74重组的重组TFIIF结合。RAP30和RPB5的中心部分对结合至关重要。通过聚类扫描，发现RAP30的点丙氨酸取代突变体Y124和Q131两个残基对rpb5的结合至关重要。内源性pol II被招募到由野生RAP30组成的TFIIF中，而不是在aa124或aa131位点上突变的RAP30。2)我们发现RPB5保留了与双链DNA结合的能力。通过扫描暴露区域的丙氨酸取代突变体，确定了RPB5暴露区域的4个氨基酸残基对DNA结合至关重要。其中，T111和SI13将直接参与dna结合。3) RMP （rpb5介导蛋白）是HBx的功能性拮抗剂，在n端，rpb5结合区和c端区域具有卷曲结构域，负责共阻遏物活性。包含细胞质定位信号（CLS）的螺旋结构域和位于c端区域的NLS对于RMP的细胞质和核定位都很重要。4)为了分析RMP的功能，通过酵母双杂交筛选寻找RMP互作伙伴。RMP本身在体外和哺乳动物细胞中被筛选并发现与RMP相互作用。另一个被反复筛选的候选基因据报道是一种辅助抑制因子，参与DNA甲基化。相互作用的生物学结果仍有待研究。

RPB5 at the tip of the lower jaw of pol II is involved in activated transcription and interacts with basal transcription factors and Hepatitis B Virus X protein (HBx). A crystal model of pol II predicted RPB5 is close to DNA downstream to transcription start site. 1) RPB5 was found to bind to recombinant TFIIF reconstituted consisting of RAP30 and RAP74. The central parts of RAP30 and RPB5 are critical for the binding. By scanning clustered, then point alanine-substitution mutants of RAP30, two residues, Y124 and Q131, were found to be critical for the RPB5-binding. Endogenous pol II was recruited to TFIIF consisting of wild RAP30 but not mutant RAP30 at aa124 or at aa131. 2) We found that RPB5 retains ability to bind to double-stranded DNA. The 4 amino acid residues in the exposed domain of RPB5 were identified to be critical for the DNA- binding analyzed by scanning alanine substitution mutants of the exposed domain. Among them, T111 and SI13 would be directly involved in the DNA-binding. 3) RMP (RPB5-mediating protein), a functional antagonist of HBx, has a coiled-coil domain at the N-terminal, RPB5-binding region, and the C-terminal region responsible for co- repressor activity. The coiled coil domain harboring cytoplasmic localization signal (CLS) and a NLS located at the C-terminal region are both important for cytoplasmic and nuclear localization of RMP. 4) To analyze function(s) of RMP, RMP-interacting partners were searched by yeast two hybrid screening. RMP, by itself, was screened and found to interacts with RMP in vitro and in mammalian cells. Another candidate, repeatedly screened out, has been reported to be a corepressor and involved in DNA methylation. Biological outcome of the interactions remains to be examined.