Seminar: Protein priming by a domesticated reverse transcriptase

Fri, 5 Feb 2016 12:20 PM - 1:10 PM

Bioscience 1006

Protein priming by a domesticated reverse transcriptase: not for viruses only

Irina A. Yushenova, PhD

Josephine Bay Paul Center for Comparative Molecular Biology and Evolution
Marine Biological Laboratory

Reverse transcriptase-related (rvt) genes are a novel class of reverse transcriptases (RT) containing an RT domain which is only distantly related to RTs of retrotransposons or retroviruses. RVT products are encoded by a non-selfish, non-proliferating, single-copy RT host gene that has been preserved by natural selection throughout the evolutionary history of large taxonomic groups, where it exhibits patchy distribution. Besides RVT, the only other known RT-derived enzyme is the telomerase reverse transcriptase, which functions to maintain chromosome ends in eukaryotic cells.

To shed light on the biological functions of RVT gene products, we focused on their biochemical properties. The HaRVT protein cloned from a filamentous gliding bacterium, Herpetosiphon aurantiacus (class Chloroflexi), was expressed in E. coli with a C-terminal 6x His -tag, and purified by sucrose gradient centrifugation and metal-affinity chromatography. Subsequently, several versions of HaRVT carrying mutations in different functional domains were created, and their activity was examined. We also checked the NcRVT protein purified from its natural host, a model ascomycete fungus Neurospora crassa. We find that both HaRVT and NcRVT initiate polymerization by covalent attachment of dNTP to a tyrosine residue. HaRVT can polymerize short products using dNTPs in the presence of Mn2+, and tends to form multimers through its N-terminal coiled-coil domain.

On balance, RVT represents the first known case of protein-primed reverse transcriptase encoded by a non-mobile chromosomal gene. It is conceivable that this unusual ability is exploited by the host species harboring rvt genes.

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