FACTORS INFLUENCING GENETIC TRANSCRIPTION--INITIATION AND TERMINATION

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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.