FACTORS INFLUENCING GENETIC TRANSCRIPTION INITIATION AND TERMINATION

各种生物体至少有一种酶可以降解RNA- dna杂交体的RNA。到目前为止，这些核糖核酸酶H （RNases H）根据初级氨基酸序列的相似性分为两类。从我们的研究中，我们知道具有良好特征的大肠杆菌RNase HI在包括人类和小鼠在内的许多不同物种中具有同源物。我们知道这些哺乳动物蛋白在序列和功能上与酿酒酵母的RNase H1相似，除了具有RNase 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RNases H，我们从第一个已完成序列的动物（秀丽隐杆线虫）中研究了这些酶。我们发现这种生物有四个RNase h1样基因；其中一个基因产生一种典型的真核RNase H1蛋白，其双链RNA结合域附着在RNase H结构域上，而其余三个蛋白的长度不同，但都含有可识别的RNase H相关氨基酸序列。标准RNase H1和至少一种附加蛋白均表现出RNase H活性。秀丽隐杆线虫中存在多种RNase H，这表明复杂生物需要多种这种类型的酶，而较低等生物只需要一种RNase H1就能很好地生存。HIV逆转录病毒（艾滋病的病因）的复制需要一种与细胞RNase H序列和结构相似的功能性RNase H。目前正在开发针对HIV的RNase H抑制逆转录病毒生长的药物。如果这些药物也与细胞酶相互作用，那么了解细胞中rnase H的数量以及抑制它们的作用的后果是很重要的。尽管药物可以有效地抑制逆转录病毒的繁殖，但它们也可能给正常的细胞过程带来问题。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研究了这些核酸的识别。在体外，检测HIV酶与各种底物的结合，揭示了各种双链和不同酶之间复杂而有趣的差异。RNA-DNA杂交体是不对称的，修饰双链的一端会强化这种不对称。HIV酶对RNA-DNA杂交体末端的修饰特别敏感，并且对两端修饰的RNA-DNA双链表现出不同的结合特性。在体内，RNA-DNA双工发生在DNA合成过程中，我们已经在酵母系统中表明，rnase H在DNA新拷贝的合成和受损DNA的修复中都很重要。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.