FACTORS INFLUENCING GENETIC TRANSCRIPTION--INITIATION AND TERMINATION

核糖核酸酶H(RNase H)在人乳头瘤病毒复制中起关键作用 逆转录病毒，包括人类免疫缺陷病毒(艾滋病毒)以及 参与正常的细胞活动，包括DNA复制。这个 这个项目的目的是为了详细了解 核糖核酸酶H底物的精确性质，即 细胞核糖核酸酶H及其多产或少产的影响 (或或多或少具有活性的RNaseH)对病毒和细胞DNA合成的影响。 我们已经发现，除了RNaseH之外，还有两种真核酶具有a 不存在的双链RNA(DsRNA)结合域 逆转录病毒或细菌RNase H与其他dsRNA结合基序不同，a DsRNA结合域的单一拷贝是必要的，也是充分的 与dsRNA结合。对于这些RNase H中的一个，我们发现每个 结构域可以独立发挥作用，但当每个结构域都在相同的多肽上时 域对其他域的属性有相当戏剧性的影响 域。RNase H与dsRNA结合的这种意外关联 区域提示DNA和蛋白质合成之间可能存在偶联 (后者已知受dsRNA影响)。 我们已经在细胞中表达了细菌RNaseH，其方式是 在酵母中被合并到逆转录病毒样颗粒中。目的 这组实验的目的是更好地理解这两种类型的 核糖核酸酶H(逆转录病毒和细菌)的不同之处，第二，看看是什么 阶段越强的细菌酶可能会抑制细菌的繁殖 病毒样颗粒。我们观察到一种相当强的抑制作用 逆转录病毒DNA从一个位置移动到另一个位置的能力 (换位)。具有较强作用的细菌核糖核酸酶H 病毒颗粒似乎干扰了病毒DNA的复制 在粒子内部。 DNA拓扑也是与RNaseH有关的一个特别重要的问题。 在转录过程中，RNA-DNA杂交体可以形成并取代 形成R环的DNA链中的一条。从理论上讲，这样的R环也可以 为DNA制造拓扑问题。我们已经证明，有 DNA拓扑异构酶I、DNA旋转酶和核糖核酸酶H之间的某些相互作用 细菌细胞控制拓扑“超螺旋”的数量 DNA

Ribonuclease H (RNase H) plays crucial roles in replication of retroviruses, including the human immunodeficiency virus (HIV) as well as involvement in normal cellular events including DNA replication. The purpose of this project is to gain a detailed understanding of the precise nature of the substrates for RNases H, the chemical makeup of cellular RNases H and the effect of either producing more or less RNase H (or a more or less active RNase H) on viral and cellular DNA synthesis. We have found that two eukaryotic enzymes have, in addition to RNase H, a double-stranded RNA (dsRNA) binding domain that is not present on retroviral or bacterial RNases H. Unlike other dsRNA-binding motifs, a single copy of the dsRNA-binding domain is necessary and sufficient for binding to dsRNA. For one of these RNases H, we have found that each domain can function independently but when on the same polypeptide each domain has rather dramatic influences on the properties of the other domain. This unexpected association of RNases H with a dsRNA-binding region suggests a possible coupling between DNA and protein synthesis (the latter of which is known to be affected by dsRNA). We have expressed bacterial RNase H in cells in such a manner that it gets incorporated into retrovirus-like particles in yeast. The purpose of this set of experiments is to better understand how the two types of RNases H (retroviral and bacterial) differ and secondly, to see at what stage the more potent bacterial enzyme might inhibit propagation of the virus-like particle. We have observed a rather strong inhibition of the ability of the retroviral DNA to move from one site to another (transposition). Having the stronger acting bacterial RNase H in the viral particle appears to interfere with replication of the viral DNA within the particle. DNA topology is also a particularly important problem related to RNase H. During the course of transcription, RNA-DNA hybrids can form and displace one of the DNA strands forming R-loops. Such R-loops, in theory can also create topological problems for the DNA. We have shown that there is some interaction between DNA topoisomerase I, DNA gyrase and RNase H in bacterial cells to control the amount of topological "super-coiling" of DNA.