Kusser I, Lowing C, Rathlef C, Köpke A K, Matheson A T
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, V8W 3P6, Canada.
Arch Biochem Biophys. 1999 May 15;365(2):254-61. doi: 10.1006/abbi.1999.1154.
A series of mutant L12 ribosomal proteins was prepared by site-directed mutations in the L12 protein gene of the archaeon Sulfolobus acidocaldarius. The mutant protein genes were overexpressed in Escherichia coli, and the products purified and incorporated into ribosomal cores which had been ethanol extracted to remove wild-type L12 protein. Measurements were made to determine if the mutation affected the binding of the L12 protein to the ribosome core or affected the translational activity of the resulting ribosome. Changing tyrosine [3] or tyrosine [5], conserved in all archaea and present in all eukarya in positions [3] and [7], to phenylalanine had no effect on binding or translational activity while changes to glycine significantly reduced binding and translational activity. Changing the single arginine [37] residue, conserved in almost all archaeal and eukaryal L12 proteins, to lysine, glutamic acid, glutamine, or glycine had no effect on binding to the core and had little or no significant effect on translational activity. The same was true when lysine [39], conserved in all archaeal L12 proteins, was changed to arginine, glutamic acid, glutamine, or glycine. Changing phenylalanine [104], the penultimate amino acid at the C-terminal end, which is conserved in all archaeal and eukaryal L12 proteins, to tyrosine or glycine had no effect on binding but lowered the translational activity by 60 and 75%, respectively, suggesting that this amino acid plays an important role in translation. Deletion of the highly charged region in the C-terminal domain, which is present in all archaeal and eukaryal L12 proteins, decreased transitional activity by 50%, suggesting this region is also involved in factor interactions.
通过对嗜热栖热放线菌L12蛋白基因进行定点突变，制备了一系列突变型L12核糖体蛋白。突变蛋白基因在大肠杆菌中过表达，产物经纯化后整合到已用乙醇提取以去除野生型L12蛋白的核糖体核心中。进行测量以确定突变是否影响L12蛋白与核糖体核心的结合，或影响所得核糖体的翻译活性。将所有古菌中保守且在所有真核生物的第[3]和[7]位存在的酪氨酸[3]或酪氨酸[5]替换为苯丙氨酸，对结合或翻译活性没有影响，而替换为甘氨酸则显著降低结合和翻译活性。将几乎所有古菌和真核生物L12蛋白中保守的单个精氨酸[37]残基替换为赖氨酸、谷氨酸、谷氨酰胺或甘氨酸，对与核心的结合没有影响，对翻译活性几乎没有或没有显著影响。当所有古菌L12蛋白中保守的赖氨酸[39]替换为精氨酸、谷氨酸、谷氨酰胺或甘氨酸时，情况也是如此。将所有古菌和真核生物L12蛋白中保守的C末端倒数第二个氨基酸苯丙氨酸[104]替换为酪氨酸或甘氨酸，对结合没有影响，但分别使翻译活性降低了60%和75%，这表明该氨基酸在翻译中起重要作用。删除所有古菌和真核生物L12蛋白中存在的C末端结构域中的高电荷区域，使过渡活性降低了50%，这表明该区域也参与因子相互作用。
