Voeller D M, Changchien L M, Maley G F, Maley F, Takechi T, Turner R E, Montfort W R, Allegra C J, Chu E
NCI-Navy Medical Oncology Branch, Bethesda, MD 20889-5105, USA.
Nucleic Acids Res. 1995 Mar 11;23(5):869-75. doi: 10.1093/nar/23.5.869.
Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.
先前的研究表明，人类TS mRNA的翻译受负向自调控机制控制。在本研究中，RNA电泳凝胶迁移率变动分析证实了大肠杆菌（E.coli）TS蛋白与其自身的大肠杆菌TS mRNA之间存在直接相互作用。在大肠杆菌TS mRNA蛋白编码区鉴定出两个顺式作用序列，一个位点对应于核苷酸207 - 460，第二个位点对应于核苷酸461 - 807。这些mRNA序列中的每一个与TS结合的相对亲和力都与全长大肠杆菌TS mRNA序列相似（IC50 = 1 nM）。还鉴定出第三个结合位点，对应于核苷酸808 - 1015，尽管其对TS的相对亲和力（IC50 = 5.1 nM）低于其他两个顺式作用元件。核苷酸结合区域内氨基酸发生突变的大肠杆菌TS蛋白保留了结合RNA的能力，而在核苷酸活性位点半胱氨酸（C146S）或叶酸结合区域内氨基酸发生突变的蛋白则无法结合TS mRNA。这些研究表明，由叶酸结合位点和/或关键半胱氨酸巯基定义的大肠杆菌TS上的区域可能代表重要的RNA结合结构域。进一步的证据表明，与TS的直接相互作用导致大肠杆菌TS mRNA翻译的体外抑制。
