ROLE OF TRNA IN AP4A SYNTHESIS BY LYS-TRNA SYNTHESIS

最近的兴趣集中在腺苷5‘，5’‘-P1-P4-的作用上。 四磷酸(Ap4A)在细胞分裂中的作用。这种分子明显是 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也与细胞 分裂，似乎是细胞横跨G1并进入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在体外通过赖氨酰-tRNA合成酶表达。 我们将对6个真核tRNALys等位受体和 与氨基酰化和Ap4A合成活性相关。在场或 缺少两个修饰的核苷导致Ap4A基因差异7倍 合成，但测定时氨基酰化活性无差异 在相同条件下纯化赖氨酰-tRNA合成酶。纯净的 TRNA-4-Lys将在特定的位置进行化学修饰，以测试 合成酶在两种氨基酰化反应中识别的结构要求 和Ap4A的合成。 2.5MU锌和tRNA存在下Ap4A的合成机制 将由反应动力学、结合研究和特定的 交换反应，并与80微米锌的交换反应机理进行比较 不需要tRNA的存在。还将比较以下几种情况 游离态和聚集态合成酶的活性。

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.