FACTORS INFLUENCING GENETIC TRANSCRIPTION INITIATION AND TERMINATION

各种生物体都至少有一种降解RNA-DNA杂交体的RNA的酶。 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。为了深入了解高等真核生物的核糖核酸酶H，我们研究了第一个完成序列的动物（秀丽隐杆线虫）的这些酶。我们发现这种生物体有四个RNase H1样基因;一个基因产生一个典型的真核RNase H1蛋白质，其双链RNA结合结构域连接到RNase H结构域，而其余三个蛋白质长度不同，但都含有可识别的RNase H相关氨基酸序列。 标准RNA酶H1和至少一种另外的蛋白质都表现出RNA酶H活性。在C. elegans提出，复杂的生物体需要几种这种类型的酶，而低等生物体只需要一种RNase H1就可以很好地生存。 HIV逆转录病毒（AIDS的起因）的复制需要功能性RNA酶H，其在序列和结构上与细胞RNA酶H相似。 目前正在开发针对艾滋病毒核糖核酸酶H的药物，以抑制逆转录病毒的生长。 如果这些药物也与细胞酶相互作用，那么了解细胞中RNase H的数量以及抑制其作用的后果就很重要了。尽管药物可以有效地抑制逆转录病毒的传播，但它们也可能对正常的细胞过程造成问题。因为这些酶识别和切割RNA-DNA杂交体的RNA，我们已经在体外和体内检测了这些核酸的识别。 在体外，检查协会的HIV酶与各种基板揭示了复杂和有趣的差异之间的各种双链体以及不同的酶。 RNA-DNA杂交体是不对称的，修饰双链体的一端会加重这种不对称性。 HIV酶对RNA-DNA杂交体末端的修饰特别敏感，并对两端修饰的RNA-DNA双链体表现出不同的结合特性。体内RNA-DNA双链体发生在DNA合成过程中，我们已经在酵母系统中表明，RNA酶H在合成新的DNA拷贝以及修复受损DNA中都很重要。 这些结果有助于我们了解这些重要蛋白的作用机制。

Various organisms have at least one enzyme that degrades the RNA of RNA-DNA hybrids. 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. To gain insight into the RNases H of higher eukaryotes, we have studied these enzymes from the first animal whose sequence has been completed (Caenorhabditis elegans). We find this organism has four RNase H1-like genes; one gene produces a protein typical of eukaryotic RNase H1 with a double-stranded RNA binding domain attached to the RNase H domain while the three remaining proteins vary in length but all contain a recognizable RNase H- related amino acid sequence. Both the standard RNase H1 and at least one of the additional proteins exhibit RNase H activity. The presence of multiple RNases H in C. elegans suggests that complex organisms require several enzymes of this type whereas lower organisms can survive well with only one RNase H1. Replication of the HIV retrovirus (the cause of AIDS) requires a functional RNase H that is similar in sequence and structure to cellular RNases H. Drugs are being developed that target RNase H of HIV to inhibit retroviral growth. If these drugs also interact with cellular enzymes, it is important to know the number of RNases H in cells as well as the consequences of inhibiting their actions. Even though drugs may effectively inhibit retroviral propagation, they might also create problems for normal cellular processes. Because these enzymes recognize and cleave RNA of RNA-DNA hybrids, we have examined recognition of such nucleic acids in vitro and in vivo. In vitro, examination of association of the HIV enzyme with various substrates reveals complex and interesting differences among the various duplexes as well as different enzymes. RNA-DNA hybrids are asymmetric and modifying one end of the duplex accents this asymmetry. The HIV enzyme is particularly sensitive to modifications at the termini of the RNA-DNA hybrid and exhibits different binding properties for RNA-DNA duplexes modified at each of the two ends. In vivo RNA-DNA duplexes occur during DNA synthesis, and we have shown in the yeast system that RNases H are important in both synthesis of new copies of DNA as well as in repair of damaged DNA. These results help us to understand the roles mechanism of action of these important proteins.