Contribution of the N-terminal Domain of Human Lysyl-tRNA Synthetase to tRNALys Binding

Jacob Calhoun


In addition to the canonical function of charging tRNALys with lysine, Lysyl-tRNA synthetase (LysRS) associates with the HIV-1 proteins Gag and Gag-Pol in a process that enables selective incorporation of tRNALys3 into budding HIV-1 virions (1), where tRNALys3 primes reverse transcription upon infection by HIV.  Mutant LysRS studies have shown tRNALys is incorporated significantly less in LysRS mutants lacking the N-terminal domain, demonstrating the N-terminal domain’s contribution to tRNALys binding as well as apparent cooperative binding of the synthetase’s domains (2, 3). The goal of this research is to quantitate the affinity of the N-terminal domain for tRNALys variants.  This is accomplished by cloning, over-expression and purification of recombinant forms of the N-terminal domain of human LysRS in E. coli (BL21 DE3).  Following the isolation of the N-terminal domain of LysRS, we demonstrated by comparative NMR studies that this N-terminal domain is primarily unstructured in the absence of RNA (3). We wish to quantify the N-terminal domain’s affinity for tRNALys3 and derivatives, using techniques such as EMSA and FA. The results of these binding studies and our understanding of how this domain influences RNA binding by LysRS will be presented and discussed.


N-terminal Domain (NTD); tRNA; Fluorescence Anisotropy (FA)

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