Yoshida Biological Laboratory Inc., Biological Information Research, Yoshida Biological Laboratory, 11-1 Takehanasotoda-cho, Yamashina-ku, Kyoto 607-8081, Japan.
Int J Mol Sci. 2023 Feb 8;24(4):3445. doi: 10.3390/ijms24043445.
Ribosomal protein bL31 in was initially detected as a short form (62 amino acids) using Kaltschmidt and Wittmann's two-dimensional polyacrylamide gel electrophoresis (2D PAGE), but the intact form (70 amino acids) was subsequently identified by means of Wada's improved radical-free and highly reducing (RFHR) 2D PAGE, which was consistent with the analysis of its encoding gene . Ribosomes routinely prepared from the K12 wild-type strain contained both forms of bL31. Δ cells, which lack protease 7, only contained intact bL31, suggesting that protease 7 cleaves intact bL31 and generates short bL31 during ribosome preparation from wild-type cells. Intact bL31 was required for subunit association, and its eight cleaved C-terminal amino acids contributed to this function. 70S ribosomes protected bL31 from cleavage by protease 7, but free 50S did not. In vitro translation was assayed using three systems. The translational activities of wild-type and Δ ribosomes were 20% and 40% lower than those of Δ ribosomes, which contained one copy of intact bL31. The deletion of bL31 reduces cell growth. A structural analysis predicted that bL31 spans the 30S and 50S subunits, consistent with its functions in 70S association and translation. It is important to re-analyze in vitro translation with ribosomes containing only intact bL31.
核糖体蛋白 bL31 在 中最初被 Kaltschmidt 和 Wittmann 的二维聚丙烯酰胺凝胶电泳 (2D PAGE) 检测为短形式（62 个氨基酸），但随后通过 Wada 的改良无自由基和高度还原 (RFHR) 2D PAGE 鉴定出完整形式（70 个氨基酸），这与对其编码基因的分析一致。从 K12 野生型菌株常规制备的核糖体含有两种形式的 bL31。缺乏蛋白酶 7 的 Δ 细胞仅含有完整的 bL31，表明蛋白酶 7 在从野生型细胞制备核糖体时切割完整的 bL31 并生成短 bL31。完整的 bL31 是亚基结合所必需的，其八个切割的 C 末端氨基酸有助于该功能。70S 核糖体保护 bL31 免受蛋白酶 7 的切割，但游离的 50S 则不能。体外翻译使用三个系统进行测定。野生型和 Δ 核糖体的翻译活性比含有一个完整 bL31 的 Δ 核糖体低 20%和 40%。bL31 的缺失降低了细胞生长。结构分析预测 bL31 跨越 30S 和 50S 亚基，与其在 70S 结合和翻译中的功能一致。用仅含有完整 bL31 的核糖体重新分析体外翻译非常重要。
