Remacha M, Jimenez-Diaz A, Bermejo B, Rodriguez-Gabriel M A, Guarinos E, Ballesta J P
Centro de Biologia Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Mol Cell Biol. 1995 Sep;15(9):4754-62. doi: 10.1128/MCB.15.9.4754.
Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently.
已获得酿酒酵母菌株,这些菌株要么有三个编码该物种中存在的四种酸性核糖体磷蛋白YP1α、YP1β、YP2α和YP2β的基因失活(三重破坏株),要么有四个基因失活(四重破坏株)。三重破坏株的核糖体,显然还有四重破坏株的核糖体,都没有结合的P蛋白。所有破坏株都能存活;然而,它们表现出冷敏感表型,在23摄氏度时生长非常缓慢。四重破坏株的细胞提取物在蛋白质合成测定中的活性约为对照的30%,并且通过添加游离酸性P蛋白可被刺激。缺乏酸性蛋白的菌株没有比亲本菌株更高的抑制活性,并且源自四重破坏株的细胞提取物没有表现出更高程度的错读,这表明酸性蛋白的缺失不会影响核糖体的准确性。然而,颗粒中缺乏P蛋白会影响细胞以及无细胞蛋白质系统中表达的蛋白质模式;向细胞提取物中添加外源P蛋白后可恢复野生型模式。此外,携带P蛋白缺陷核糖体的菌株无法形成孢子,但在用缺失基因之一进行转化后可恢复这种能力。这些结果表明,酸性蛋白不是蛋白质合成的绝对必需物质,但可调节60S亚基的活性,对某些mRNA的翻译有不同影响。