Factors Influencing Genetic Transcription Initiation

各种生物体都至少有一种降解RNA-DNA杂交体的RNA的酶。这种杂合体在体内由转录产生，并且通常与DNA复制相关，甚至在逆转录病毒（包括HIV）的复制中。到目前为止，这些核糖核酸酶H（RNases H）根据一级氨基酸序列相似性分为两类。从我们的研究中，我们知道，充分表征的大肠杆菌RNase HI在许多不同的物种，包括人类和小鼠中具有同源物。我们知道，这些哺乳动物蛋白质在序列和功能上类似于酿酒酵母的RNA酶H1，除了RNA酶H活性外，还具有双链RNA结合活性。类似地，细菌RNase HII蛋白在真核生物中具有对应物。RNA酶H执行重要的细胞功能，重要的是要知道这些酶是如何合成的。例如，在基于阿糖胞苷的反义疗法中使用DNA药物依赖于内源性RNA酶H来降解某些引起疾病的mRNA（在不适当的时间或位置合成的RNA）。提高靶细胞中RNase H活性的能力可以使这些反义DNA更有效的药物。类似地，某些类型的靶向抑制HIV逆转录酶的RNase H活性的药物也可能抑制细胞酶，导致不期望的效果。从S.酿酒酵母具有高水平的RNase H活性，但在E.大肠杆菌无活性。我们发现其他多肽与活性形式的酶共纯化，表明多个亚基包含活性酶。RNH 2A基因表达的细胞周期调节以及由于重叠DNA位点引起的差异表达表明蛋白质在细胞周期的不同阶段可能具有不同的亚基以参与DNA复制或DNA修复。这些结果表明RNase H2 Ap的诱导或表达可能不足以增加与该多肽相关的RNase H活性。事实上，当RNase H2 Ap确实发生过表达时，我们发现RNase H活性仅略有增加。我们还发现人和小鼠的RNase H1蛋白在底物存在下形成二聚体，并且这种复合物导致酶的持续合成能力（即，该蛋白倾向于在释放和攻击第二个分子之前降解一个大小无关的底物分子）。今年的这些结果有助于我们了解这些重要蛋白质的作用机制

Various organisms have at least one enzyme that degrades the RNA of RNA-DNA hybrids. Such hybrids result in vivo from transcription and often are associated with DNA replication, even in replication of retroviruses including HIV. These ribonucleases H (RNases H), so far, fall into two classes based upon primary amino acid sequence similarity. From our studies, we know that the well-characterized Escherichia coli RNase HI has homologs in many different species including human and mouse. We know that these mammalian proteins resemble in sequence and function the RNase H1 of Saccharomyces cerevisiae by having a double-stranded RNA-binding activity in addition to the RNase H activity. Similarly, the bacterial RNase HII protein has counterparts in eukaryotes. RNases H perform important cellular functions and it is important to know how and by what means these enzymes are synthesized. For example, the use of DNA drugs employed in oligonucleotide-based antisense therapy relies on endogenous RNases H to degrade certain disease causing mRNAs (RNAs synthesized at inappropriate times or locations). The ability to increase RNase H activity in target cells could make these antisense DNAs more effective drugs. In a similar vein, certain types of drugs targeted to inhibit the RNase H activity of HIV reverse transcriptase could also inhibit the cellular enzymes leading to undesired effects. The RNase H2Ap protein purified from S. cerevisiae has high levels of RNase H activity yet the same protein expressed in E. coli is inactive. We find other polypeptides co-purify with the active form of the enzyme suggesting that multiple subunits comprises the active enzyme. Cell-cycle regulation of the RNH2A gene expression together with the differential expression due to the overlapping DNA sites suggests the protein may have different subunits at different stages of the cell-cycle to participate in either DNA replication or DNA repair. These results indicate induction or expression of the RNase H2Ap may not be sufficient for increasing RNase H activity associated with this polypeptide. In fact, when overexpression of the RNase H2Ap does occur, we have found only a modest increase in RNase H activity. We have also found that the RNase H1 protein of human and mouse form a dimer in the presence of substrate and that this complex leads to processivity of the enzyme (i.e., the protein tends to degrade one molecule of substrate regradless of size before releasing and attacking a second molecule). These results from this year help us to understand the roles mechanism of action of these important proteins