The N-terminal domain of mammalian lysyl-tRNA synthetase is a functional tRNA-binding domain
The N-terminal domain of mammalian lysyl-tRNA synthetase is a functional tRNA-binding domain
复制标题
DOI：
10.1074/jbc.m109759200
复制
发表时间：
2002-01-18
影响因子：
4.8
通讯作者：
Mirande, M
中科院分区：
文献类型：
--
作者：
Francin, M;Kaminska, M;Mirande, M
来自高等真核生物的赖氨酸- trna合成酶具有一个富含赖氨酸的n端多肽延伸，附加在经典的原核类LysRS结构域上。带移分析表明，这个额外的结构域使LysRS具有非特异性tRNA结合特性。与天然酶相比，LysRS的n端截断衍生物LysRS- deltan在氨基酰化反应中对tRNA(3)（Lys）的表观亲和力降低了100倍，对tRNA(3)（Lys）的K-m增加了3倍。分离的LysRS的n结构域对tRNA也表现出较弱的亲和力，这表明LysRS的催化结构域和n结构域协同作用，为tRNA提供了高亲和力的结合位点。更详细的分析表明，LysRS结合并特异性地使一个模仿tRNA 3的氨基酸受体茎环结构的RNA微螺旋氨基酸酰化，而LysRS- deltan则没有。因此，将一个额外的rna结合结构域合并到细菌样的LysRS中可以提高酶的催化效率，特别是在体内普遍存在低浓度脱酰基tRNA的情况下。我们的研究结果为tRNA（Lys）在真核细胞中的通道提供了新的见解，并揭示了天然LysRS触发tRNA(3)（Lys）包装到人类免疫缺陷病毒I型病毒颗粒中的可能需求。
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, 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 I viral particles.