Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5
Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5
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DOI：
10.1261/rna.688207
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发表时间：
2007-12-01
期刊：
影响因子：
4.5
通讯作者：
Komar, Anton A.
中科院分区：
文献类型：
--
作者：
Galkin, Oleksandr;Bentley, Amber A.;Komar, Anton A.
核糖体蛋白 (rp) S5 属于核糖体蛋白家族，其中包括细菌 rpS7。 rpS5 构成 40S 核糖体亚基上的出口 (E) 位点的一部分，对于酵母的活力至关重要。人类 rpS5 与酿酒酵母 rpS5 67% 相同，79% 相似，但缺乏真菌中存在的带负电荷（pI；类似于 3.27）的 21 个氨基酸长 N 端延伸。在这里，我们报告用人类同源物替换酵母 rpS5 产生了一种可行的酵母菌株，其生长速度降低了 20%-25%。这种替换还导致突变株中重多核糖体成分适度增加，表明存在翻译延伸或终止缺陷，并导致延伸因子 eEF3 和 eEF1A 的多核糖体关联减少。此外，突变株的特征是+1和-1程序移码适度增加以及UAA终止密码子的超准确识别。突变株中蟋蟀麻痹病毒 (CrPV) IRES 和两种哺乳动物细胞 IRES（CAT-1 和 SNAT-2）的活性也有所增加。一致地，rpS5 替换导致 CrPV IRES 和突变酵母核糖体之间的直接相互作用增强。总而言之，这些数据表明 rpS5 在维持真核生物翻译的准确性方面发挥着重要作用，并表明酵母 rpS5 的带负电荷的 N 端延伸可能会影响特定 mRNA 的核糖体募集。
Ribosomal protein ( rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit ( E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI; similar to 3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%-25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and -1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.