Incorporation of lysyl-tRNA synthetase into human immunodeficiency virus type 1.

During human immunodeficiency virus type 1 (HIV-1) assembly, tRNA(Lys) isoacceptors are selectively incorporated into virions and tRNA(Lys)3 is used as the primer for reverse transcription. We show herein that the tRNA(Lys)-binding protein, lysyl-tRNA synthetase (LysRS), is also selectively packaged into HIV-1. The viral precursor protein Pr55gag alone will package LysRS into Pr55gag particles, independently of tRNA(Lys). With the additional presence of the viral precursor protein Pr160gag-pol, tRNA(Lys) and LysRS are both packaged into the particle. While the predominant cytoplasmic LysRS has an apparent M(r) of 70,000, viral LysRS associated with tRNA(Lys) packaging is shorter, with an apparent M(r) of 63,000. The truncation occurs independently of viral protease and might be required to facilitate interactions involved in the selective packaging and genomic placement of primer tRNA.

Efficient aminoacylation of tRNA(Lys,3) by human lysyl-tRNA synthetase is dependent on covalent continuity between the acceptor stem and the anticodon domain.

In this work, we probe the role of the anticodon in tRNA recognition by human lysyl-tRNA synthetase (hLysRS). Large decreases in aminoacylation efficiency are observed upon mutagenesis of anticodon positions U35 and U36 of human tRNA(Lys,3). A minihelix derived from the acceptor-TPsiC stem-loop domain of human tRNA(Lys,3)was not specifically aminoacylated by the human enzyme. The presence of an anticodon-derived stem-loop failed to stimulate aminoacylation of the minihelix. Thus, covalent continuity between the acceptor stem and anticodon domains appears to be an important requirement for efficient charging by hLysRS. To further examine the mechanism of communication between the critical anticodon recognition elements and the catalytic site, a two piece semi-synthetic tRNA(Lys, 3)construct was used. The wild-type semi-synthetic tRNA contained a break in the phosphodiester backbone in the D loop and was an efficient substrate for hLysRS. In contrast, a truncated variant that lacked nucleotides 8-17 in the D stem-loop displayedseverely reduced catalytic efficiency. The elimination of key tRNA tertiary structural elements has little effect on anticodon-dependent substrate binding but severely impacts formation of the proper transition state for catalysis. Taken together, our studies provide new insights into human tRNA structural requirements for effective transmission of the anticodon recognition signal to the distal acceptor stem domain.

Human lysyl-tRNA synthetase accepts nucleotide 73 variants and rescues Escherichia coli double-defective mutant.

The nucleotide 73 (N73) "discriminator" base in the acceptor stem is a key element for efficient and specific aminoacylation of tRNAs and of microhelix substrates derived from tRNA acceptor stems. This nucleotide was possibly one of the first to be used for differentiating among groups of early RNA substrates by tRNA synthetases. In contrast to many other synthetases, we report here that the class II human lysyl-tRNA synthetase is relatively insensitive to the nature of N73. We cloned, sequenced, and expressed the enzyme, which is a close homologue of the class II yeast aspartyl-tRNA synthetase whose co-crystal structure (with tRNAAsp) is known. The latter enzyme has a strong requirement for G73, which interacts with 4 of the 14 residues within the "motif 2" loop of the enzyme. Even though eukaryotic lysine tRNAs also encode G73, the motif 2 loop sequence of lysyl-tRNA synthetase differs at multiple positions from that of the aspartate enzyme. Indeed, the recombinant human lysine enzyme shows little preference for G, and even charges human tRNA transcripts encoding the A73 found in E. coli lysine tRNAs. Moreover, while the lysine enzyme is the only one in E. coli to be encoded by two separate genes, a double mutant that disables both genes is complemented by a cDNA expressing the human protein. Thus, the sequence of the loop of motif 2 of human lysyl-tRNA synthetase specifies a structural variation that accommodates nucleotide degeneracy at position 73. This sequence might be used as a starting point for obtaining highly specific interactions with any given N73 by simple amino acid replacements.