X-ray crystallographic studies of the Moloney murine leukemia virus reverse transcriptase

Reverse transcriptases (RTs) are essential multi-domain enzymes that convert the single-stranded RNA genome of retroviruses into double-stranded DNA for integration into the host chromosome by coupling an RNA- and DNA-dependent DNA polymerase activity with a ribonuclease (RNase) H function. Despite architectural differences, RTs from different viruses exhibit similar biochemistry but differ in processivity, fidelity and oligomeric state. Crystallographic studies on these enzymes have involved the Human immunodeficiency virus type 1 (HIV-1) RT, a key target in anti-AIDS therapies, the HIV-2 RT and the N-terminal fragment of Moloney murine leukemia virus (MMLV) RT. This dissertation focuses on determining, for the first time, crystal structures of the full-length MMLV RT to understand structure-function relationships in reverse transcriptases. Part I provides a brief introduction to x-ray crystallography and the biology of reverse transcriptases. Part II describes how the solubility of the MMLV RT was improved to result in crystals suitable for a crystal structure determination. Part III discusses the determination of the 3.0 A crystal structure of the MMLV RT complexed to DNA and the implications from the structure. Striking contrasts are revealed in the structures of MMLV RT and HIV-1 RT based on differences in relative positions of the different domains in these enzymes. Part IV describes the crystal structure at 7.0 A of a "quarternary" complex of MMLV RT and confirms the flexibility of the RNase H domain that can "swing around" relative to the polymerase portion. The DNA duplex is packed end-to-end in the crystal lattice. These differences might explain the structural basis for the functional differences between the MMLV RT and HIV-1 RT, including its ability to perform processive DNA synthesis in a monomeric form. Part V is on previously unreported sequence similarities and conservation of residues in the thumb and connection domains of MMLV RT with other RTs and concludes that there may be residues in these domains that play important roles in protein-DNA interactions during reverse transcription and that the crystal structure of MMLV RT can serve as a model to drive biochemical studies on other reverse transcriptases.