Student Profle: Shawn Rumrill

Research Summary
Reverse transcription is a remarkable process by which RNA is copied into a double-stranded (ds)DNA, the products of which make up an estimated 8% of the human genome via retrotransposons and endogenous retroviruses. The reverse transcriptase enzyme (RT) is a major target of HIV treatment, accounting for more than half of the drugs on the market. Understanding RT’s mechanism of action is a rigorous test of our knowledge of enzymes and the central dogma of biology. While HIV-1 reverse transcriptase (RT) has been characterized both structurally and biochemically in depth, the initial stages of reverse transcription are still a mystery. This provides an exciting opportunity for me to resolve, in atomic detail, how reverse transcription occurs. During initiation, RT binds an 18-nucleotide (nt) primer-binding sequence (PBS) duplex formed by a primer tRNA and viral (v)RNA complex and incorporates 6-9 nts in an exceedingly slow manner before somehow progressing to a rapid and processive elongation stage. Very recently, the first snapshots of RT initiation complexes (RTIC) have been published. Amazingly, a crystal structure details an RTIC poised for incorporation of the first dNTP into the dsRNA, while a cryo-EM structure captures an RTIC right after incorporation of the first dNTP. The two structures are complementary: the cryo-EM structure visualizes the RNA elements protruding outside of the RT nucleic acid-binding cleft, while the crystal structure maintains a higher resolution for the RT/dsRNA core and interface. Both structures correspond to catalytically inefficient conformations with a hyperextended thumb subdomain and a distorted polymerase active site conformation, perhaps explaining the very slow initial incorporation rate. To understand the structural and dynamic changes in RT from initiation to elongation, I will use X-ray crystallographic and cryo-EM approaches to capture successive single nucleotide incorporation states of RT/vRNA/tRNA.