The N-terminal domain of mammalian Lysyl-tRNA synthetase is a functional tRNA-binding domain.
Lysyl-tRNA synthetase from higher eukaryotes possesses a lysine-rich N-terminal polypeptide extension appended to a classical prokaryotic-like LysRS domain. Band shift analysis showed that this extra domain provides LysRS with nonspecific tRNA binding properties. A N-terminally truncated derivative of LysRS, LysRS-DeltaN, displayed a 100-fold lower apparent affinity for tRNA(3)Lys and a 3-fold increase in K(m) for tRNA(3)Lys in the aminoacylation reaction, as compared with the native enzyme. The isolated N-domain of LysRS also displayed weak affinity for tRNA, suggesting that the catalytic and N-domains of LysRS act synergistically to provide a high affinity binding site for tRNA. A more detailed analysis revealed that LysRS binds and specifically aminoacylates an RNA minihelix mimicking the amino acid acceptor stem-loop structure of tRNA(3)Lys, whereas LysRS-DeltaN did not. As a consequence, merging an additional RNA-binding domain into a bacterial-like LysRS increases the catalytic efficiency of the enzyme, especially at the low concentration of deacylated tRNA prevailing in vivo. Our results provide new insights into tRNA(Lys) channeling in eukaryotic cells and shed new light on the possible requirement of native LysRS for triggering tRNA(3)Lys packaging into human immunodeficiency virus, type 1 viral particles.
Francin
Mathilde
M
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette, France.
Kaminska
Monika
M
Kerjan
Pierre
P
Mirande
Marc
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D006224:Cricetinae; D017931:DNA Primers; D008250:Lysine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D012357:RNA, Transfer, Lys / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D012357:RNA, Transfer, Lys; D008250:Lysine-tRNA Ligase