THYMIDYLATE SYNTHASE REGULATION

胸苷酸合酶(TS)的生物合成是新近发现的 通过将蛋白质与其自身的mRNA结合而下调，以及 这一调节参与了癌细胞的发展 对几种针对这种酶的抗代谢药物的抗药性。 因为TS-mRNA与TS结合和TS-酶活性都是 观察到随着巯基乙醇(ME)的增加而类似地增加 浓度，我们测试这些亲和力和活性是否增加 符合由单一基因控制的假说 共同氧化还原位点或结合独立控制假说 和两个不同站点的活动。因此，描述两者的数据集 TS与TS-mRNA的结合及TS的活性(无mRNA存在) 添加了ME的模型分别适用于绑定到的模型 两个已知的信使核糖核酸位点，用于胸腺嘧啶酸的产生和适当的 可能的氧化还原控制的选择。两种情况下的似然比检验 氧化还原模型显示，氧化还原参数向量非常 如果从绑定或 活动数据，从而导致这两个假说都被拒绝。 因此，TS合成的氧化还原调节似乎是复杂的， 涉及多个站点之间的耦合交互作用。此外, 一些TS-mRNA结合区的结构研究已经开始。 确定了两个可能的35聚体茎环结构的熔点 在此之前，我们检查了紫外线的吸收情况，发现 与预测的富含G-C的茎。此外，因为茎环 结构的不同之处在于含有一个游离的腺嘌呤或 进行碱基配对的1D-核磁共振以寻找FAR的证据 U-配对腺嘌呤的咪唑氢下场移位。换挡 小于13ppm，因此需要2D-核磁共振。

The biosynthesis of thymidylate synthase (TS) has recently been found to be down-regulated by binding of the protein to its own mRNA, and this regulation is involved in the development by cancer cells of resistance to several antimetabolites targeted at this enzyme. Because both TS-mRNA binding to TS and TS-enzyme activity were observed to increase similarly with increasing mercaptoethanol (ME) concentration, we tested whether these affinity and activity increases were consistent with either the hypothesis of control by a single common redox site or the hypothesis of independent control of binding and activity by two separate sites. Thus, data sets describing both the binding of TS to TS-mRNA and the activity of TS (no mRNA present) with added ME were fit separately to models accounting for binding to two known mRNA sites, for thymidylate production, and for appropriate choice of putative redox control. Likelihood ratio testing for either redox model revealed that the redox parameter vector is very significantly different when estimated from the binding or the activity data, thus leading to the rejection of both hypotheses. Hence, redox regulation of TS synthesis appears to be complex, involving coupled interactions between multiple sites. In addition, some structural investigation of the TS-mRNA binding region was begun. Melting points of two possible 35-mer stem-loop structures, identified previously, were examined for UV absorbance and found to be consistent with predicted G-C-rich stems. Furthermore, because the stem-loop structures differ in having a single adenine either free or base-paired, 1D-NMR was performed to seek evidence for far down-field-shifted imidazole hydrogens of U-paired adenine. Shifting was less than 13 ppm, thus indicating the need for 2D-NMR.