Kaminski A, Hunt S L, Patton J G, Jackson R J
Department of Biochemistry, University of Cambridge, United Kingdom.
RNA. 1995 Nov;1(9):924-38.
The requirement of PTB, polypyrimidine tract binding protein, for internal initiation of translation has been tested using an RNA affinity column to deplete rabbit reticulocyte lysates of PTB. The affinity column was prepared by coupling CNBr-activated Sepharose with the segment of the 5'-untranslated region of encephalomyocarditis virus (EMCV) RNA previously shown to bind PTB. Lysates passed through this column were devoid of PTB, and were incapable of internal initiation of translation dependent on the EMCV 5'-untranslated region, while retaining the capacity for translation dependent on ribosome scanning. Full activity for internal initiation was restored by the addition of recombinant PTB at the physiologically relevant concentration of about 5 micrograms/mL. When various PTB deletion mutants were tested, it was found that this activity required virtually the full-length protein. Thus, PTB is an essential protein for internal initiation promoted by the EMCV 5'-untranslated region. However, the PTB-depleted lysate retained the capacity for internal initiation promoted by the 5'-untranslated regions of another cardiovirus, Theiler's murine encephalomyelitis virus, and of the unrelated hepatitis C virus, and in neither case did addition of recombinant PTB stimulate internal initiation. Therefore, PTB is not a universal internal initiation factor that is indispensable in every case of internal ribosome entry.
利用RNA亲和柱去除兔网织红细胞裂解物中的多嘧啶序列结合蛋白（PTB），以检测PTB对翻译内部起始的需求。亲和柱是通过将溴化氰活化的琼脂糖与先前已证明能结合PTB的脑心肌炎病毒（EMCV）RNA 5'-非翻译区片段偶联制备而成。通过该柱的裂解物不含PTB，并且无法进行依赖于EMCV 5'-非翻译区的翻译内部起始，同时保留了依赖核糖体扫描的翻译能力。通过添加生理相关浓度约为5微克/毫升的重组PTB，可恢复内部起始的全部活性。当测试各种PTB缺失突变体时，发现该活性实际上需要全长蛋白。因此，PTB是EMCV 5'-非翻译区促进的翻译内部起始所必需的蛋白质。然而，PTB缺失的裂解物保留了另一种心病毒——泰勒氏小鼠脑脊髓炎病毒以及无关的丙型肝炎病毒5'-非翻译区促进的翻译内部起始能力，并且在这两种情况下，添加重组PTB均未刺激内部起始。因此，PTB并非在每种核糖体内部进入情况下都不可或缺的通用内部起始因子。
