Department of Life Science, College of Science, Rikkyo University, Tokyo, Japan
Research Center for Life Science, College of Science, Rikkyo University, Tokyo, Japan.
J Bacteriol. 2021 Apr 21;203(10). doi: 10.1128/JB.00599-20.
Ribosomal protein S14 can be classified into three types. The first, the C+ type has a Zn binding motif and is ancestral. The second and third are the C- short and C- long types, neither of which contain a Zn binding motif and which are ca. 90 residues and 100 residues in length, respectively. In the present study, the C+ type S14 from ribosomes (S14BsC+) were completely replaced by the heterologous C- long type of S14 from (S14Ec) or (S14Se). Surprisingly, S14Ec and S14Se were incorporated fully into 70S ribosomes in However, the growth rates as well as the sporulation efficiency of the mutants harboring heterologous S14 were significantly decreased. In these mutants, the polysome fraction was decreased and the 30S and 50S subunits accumulated unusually, indicating that cellular translational activity of these mutants was decreased. analysis showed a reduction in the translational activity of the 70S ribosome fraction purified from these mutants. The abundance of ribosomal proteins S2 and S3 in the 30S fraction in these mutants was reduced while that of S14 was not significantly decreased. It seems likely that binding of heterologous S14 changes the structure of the 30S subunit, which causes a decrease in the assembly efficiency of S2 and S3, which are located near the binding site of S14. Moreover, we found that S3 from cannot function in unless S14Se is present. S14, an essential ribosomal protein, may have evolved to adapt bacteria to zinc-limited environments by replacement of a zinc-binding motif with a zinc-independent sequence. It was expected that the bacterial ribosome would be tolerant to replacement of S14 because of the previous prediction that the spread of C- type S14 involved horizontal gene transfer. In this study, we completely replaced the C+ type of S14 in ribosome with the heterologous C- long type of S14 and characterized the resulting chimeric ribosomes. Our results suggest that the ribosome is permissive for the replacement of S14, but coevolution of S3 might be required to utilize the C- long type of S14 more effectively.
核糖体蛋白 S14 可分为三种类型。第一种是 C+ 型，具有锌结合基序，是原始的。第二种和第三种是 C-短型和 C-长型，都不含锌结合基序，分别约 90 个和 100 个氨基酸。在本研究中，核糖体中的 C+ 型 S14 完全被来自 的异源 C-长型 S14（S14Ec）或 的 S14（S14Se）取代。令人惊讶的是，S14Ec 和 S14Se 完全掺入到 70S 核糖体中，但含有异源 S14 的突变体的生长速率和孢子形成效率显著降低。在这些突变体中，多核糖体分数减少，30S 和 50S 亚基异常积累，表明这些突变体的细胞翻译活性降低。分析表明，从这些突变体中纯化的 70S 核糖体部分的翻译活性降低。这些突变体中 30S 亚基中核糖体蛋白 S2 和 S3 的丰度降低，而 S14 的丰度没有显著降低。似乎异源 S14 的结合改变了 30S 亚基的结构，导致 S2 和 S3 的组装效率降低，而 S2 和 S3 位于 S14 的结合位点附近。此外，我们发现 中的 S3 除非存在 S14Se，否则不能在 中发挥作用。S14 是一种必需的核糖体蛋白，通过用非锌依赖的序列替换锌结合基序，可能已经进化为使细菌适应锌有限的环境。人们期望细菌核糖体能够耐受 S14 的替换，因为之前的预测表明 C- 型 S14 的传播涉及水平基因转移。在本研究中，我们完全取代了 核糖体中的 C+ 型 S14，并用异源 C-长型 S14 取代，对得到的嵌合核糖体进行了表征。我们的结果表明， 核糖体允许 S14 的替换，但可能需要 S3 的共进化，以更有效地利用 C- 长型 S14。
