Initiation of nucleic acid synthesis is a critical step in the replication and transcription of genetic information. A detailed molecular description of this process will be important for research in a wide variety of fields, ranging from the design of antiviral drugs to the regulation of cancer cell proliferation. I propose to study the initiation of DNA synthesis by human immunodeficiency virus type 1 (HIV- 1) reverse transcriptase (RT) from a tRNA primer annealed to an RNA template, a project that builds on the extensive structural, biochemical, and molecular genetic information already available. Crystals of HIV-1 RT complexed with tRNA alone and with tRNA annealed to a short template RNA have been obtained in the laboratory of Dr. Thomas Steitz, and diffract to moderate resolution. To gain crystallographic experience, I will join the ongoing work to solve these structures. To complement and extend these studies, I plan to systematically alter both the protein and nucleic acid components of the initiation complex, with the goal of obtaining crystals that diffract to higher resolution. Specifically, I will (1) crystallize and determine the structure of protein-nucleic acid complexes involved in the initiation of DNA synthesis by HIV-1 RT, and (2) evaluate the functional importance of these complexes using in vitro assays for initiation. Questions that I hope to address through these studies include: What contacts are made between RT and RNA during the formation of an initiation complex? How is the tRNA primer unwound and annealed to the template RNA? What conformational changes occur in RT during the initiation of DNA synthesis? Answers to these questions will provide valuable insight into the mechanisms by which polymerases synthesize nucleic acids.
