ROLE OF TRNA IN AP4A SYNTHESIS BY LYS-TRNA SYNTHESIS

最近的兴趣集中在腺苷5 '，5“"-P1-P4-腺苷的作用上。 四磷酸（Ap 4A）参与细胞分裂。 这种分子明显 增加增殖细胞，与DNA聚合酶结合并刺激 体内DNA合成。 此外，tRNA-4-Lys与细胞凋亡有关。 分裂，似乎是细胞穿过G1进入S所必需的 相位 这两种细胞反应最近被我们的 证明tRNA-4-Lys优先诱导合成 Ap-4-A通过赖氨酰-tRNA合成酶在体外。 将在6种真核tRNALys同种受体和 与氨酰化和Ap 4A合成活性相关。 的存在或 缺乏两个修饰的核苷导致Ap 4A的7倍差异 合成，但在氨酰化活性无差异时， 纯化的赖氨酰-tRNA合成酶。 纯化 tRNA-4-Lys将在特定位点进行化学修饰，以检测 氨酰化和氨酰化中合成酶识别的结构要求 Ap 4A合成 在2.5 μ M锌和tRNA存在下Ap 4A合成的机制 将通过反应动力学、结合研究和特异性 交换反应，并与80 μ M锌的机制相比， 不需要tRNA的存在。 还将比较 游离和聚集合成酶的活性。

Recent interest has centered on the role of adenosine 5',5'''-P1-P4- tetraphosphate (Ap4A) in cell division. This molecule is markedly increased in proliferating cells, binds to DNA polymerase and stimulates DNA synthesis in vivo. Also, tRNA-4-Lys has been implicated in cell division and appears to be required for cell to transverse G1 and enter S phase. These two cell responses have recently been linkedxby our demonstration that tRNA-4-Lys preferentially induces the synthesis of Ap-4-A by lysyl-tRNA synthetase in vitro. Comparisons will be made between the 6 eucaryotic tRNALys isoacceptors and correlated to aminoacylation and Ap4A synthetic activity. The presence or absence of two modified nucleosides results in a 7-fold difference in Ap4A synthesis, but no difference in aminoacylation activity when assayed with purified lysyl-tRNA synthetase under the same conditions. Purified tRNA-4-Lys will be chemically modified at specific sites to test the structural requirements for synthetase recognition in both aminoacylation and Ap4A synthesis. The mechanism of Ap4A synthesis in the presence of 2.5 MuM zinc and tRNA will be determined by reaction kinetics, binding studies and specific exchange reactions, and compared to the mechanism at 80 MuM zinc which does not require the presence of tRNA. Comparisons will also be made between the activity of free and aggregated synthetase.