FACTORS INFLUENCING GENETIC TRANSCRIPTION--INITIATION AND TERMINATION

影响基因转录的因素——起始和终止

• 批准号: 3756619
• 负责人: R CROUCH
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3756619
• 关键词: DNA gyrase; DNA topoisomerases; Escherichia coli; RNA directed DNA polymerase; Retroviridae; bacterial genetics; double stranded RNA; enzyme mechanism; enzyme structure; genetic transcription; nucleic acid hybridization; protein structure function; ribonuclease III; transcription termination

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.

核糖核酸酶H（RNase H）在细胞复制中起着至关重要的作用。 逆转录病毒，包括人类免疫缺陷病毒（HIV）以及 参与正常细胞活动，包括DNA复制。 的 该项目的目的是获得详细的了解 RNA酶H底物的精确性质， 细胞RNase H和产生更多或更少RNase H的影响 (or或多或少活性的RNA酶H）对病毒和细胞DNA合成的影响。 我们已经发现，除了RNase H外，两种真核生物酶具有一种 双链RNA（dsRNA）结合结构域，不存在于 逆转录病毒或细菌RNA酶H。 与其他dsRNA结合基序不同， dsRNA结合结构域的单拷贝是必要的，并且足以 与dsRNA结合。 对于这些RNase H中的一种，我们已经发现， 结构域可以独立发挥作用，但当在同一多肽上时， 一个域对另一个域的性质具有相当大的影响， 域 RNA酶H与dsRNA结合蛋白的这种意想不到的关联， 该区域表明DNA和蛋白质合成之间可能存在耦合 (the已知后者受dsRNA影响）。 我们已经在细胞中表达了细菌RNase H， 被整合到酵母中的逆转录病毒样颗粒中。 目的 这组实验的目的是为了更好地理解这两种类型的 RNA酶H（逆转录病毒和细菌）不同，其次，看看在什么 阶段更有效的细菌酶可能会抑制繁殖的 病毒样颗粒 我们观察到一种相当强烈的抑制作用， 逆转录病毒DNA从一个位点移动到另一个位点的能力 （换位）。 在细菌中有作用更强的核糖核酸酶H， 病毒颗粒似乎干扰病毒DNA的复制 在粒子中。 DNA拓扑结构也是与RNase H相关的一个特别重要的问题。 在转录过程中，RNA-DNA杂合体可以形成并取代 形成R环的DNA链之一 理论上，这种R环也可以 为DNA制造拓扑问题。 我们已经证明， DNA拓扑异构酶I、DNA促旋酶和RNase H相互作用 细菌细胞来控制拓扑"超螺旋"的数量， DNA.