Relationship between structure and function of eubacterial SRP-like particle

真细菌类SRP颗粒结构与功能的关系

• 批准号: 10660072
• 负责人: NAKAMURA Kouji
• 金额: $ 2.62万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660072/
• 关键词: histone-like protein HBsu; RNA-protein interaction; protein secretion; domain structure; double-strand RNA; B. subtilis; signal recognition particle; Alu domain; 蛋白質合成; 伸長因子EF-G; シグナル認識粒子RNA; 大腸菌; RNA結合蛋白質; RNA高次機能; 真正細菌

Small cytoplasmic RNA (scRNA) is metabolically stable and abundant in Bacillus subtilis cells. Consisting of 271 nucleotides, it is structurally homologous to mammalian signal recognition particle RNA. In contrast to 4.5 S RNA of Escherichia coli, B. subtilis scRNA contains an Alu domain in addition to the evolutionarily conserved S domain. In this study, we show that a 10-kDa protein in B. subtilis cell extracts has scRNA binding activity at the Alu domain. The in vitro binding selectivity of the 10-kDa protein shows that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two loops. Purification and subsequent analyses demonstrated that the 10-kDa protein is HBsu, which was originally identified as a member of the histone-like protein family. By constructing a HBsu-deficient B. subtilis mutant, we showed that HBsu is essential for normal growth. Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA. … More Moreover, the co-precipitation of HBsu with (His)6-tagged Ffh depended on the presence of scRNA, suggesting that HBsu, Ffh, and scRNA make a ternary complex and that scRNA serves as a functional unit for binding. These results demonstrated that HBsu is the third component of a signal recognition particle-like particle in B. subtilis that can bind the Alu domain of scRNA. In Escherichia coli, 4.5S RNA is found in complexes with both protein translocation protein, Ffh (a bacterial homolog of mammalian SRP54) and protein synthesis elongation factor G (EF-G). To analyze the function of 4.5S RNA in translation, we initially assessed the sensitivity of the association of 4.5S RNA with the ribosome after treatment with antibiotics that affect various stages of protein synthesis. Fusidic acid and viomycin caused 4.5S RNA to cosediment with the 70S ribosomal fraction, indicating that 4.5S RNA enters the ribosome before ribosomal translocation and release of EF-G-GDP from the ribosome. On the other hand , depletion of 4.5S RNA led to the retention of a significant amount of EF-G on 70S ribosomes. In addition, 4.5S RNA shares a conserved decanucleotide sequence (58GAAGCAGCCA67) motif with the characterized EF-G-binding site at positions 1068-1077 on 23S RNA. We therefore examined by gel mobility-shift assay whether or not mutations in the domain-IV region of 4.5S RNA, including this conserved motif, disturb the binding of EF-G to 23S RNA. Any mutation at the C62, G64 or A67 residues within this motif abolished competition activity. Therefore, we propose that 4.5S RNA is concerned with the mode of association of EF-G with the ribosomes. Moreover, this function depends on the secondary structure of 4.5S RNA as well as a ten-base sequence conserved between the two RNAs. Less

枯草杆菌小分子胞质RNA(ScRNA)在枯草杆菌细胞中代谢稳定，含量丰富。它由271个核苷酸组成，在结构上与哺乳动物信号识别颗粒RNA同源。枯草杆菌单链RNA含有进化上保守的S结构域，而不同于大肠杆菌的4.5GyaRNA。在本研究中，我们证明了枯草杆菌细胞提取物中的10 kDa蛋白具有Alu结构域的scRNA结合活性。10-kDa蛋白的体外结合选择性表明，它识别由两个环中连续5个互补序列引起的scRNA的Alu结构域的较高结构。纯化和随后的分析表明，该10 kDa蛋白是HBsu，最初被鉴定为组蛋白样蛋白家族的成员。通过构建HBsu缺失的枯草芽孢杆菌突变体，我们证明了HBsu对正常生长是必不可少的。用抗HBsu抗体免疫沉淀细胞裂解物可产生scRNA。…此外，HBsu与(His)-6标记的FFH的共沉淀依赖于scRNA的存在，这表明HBsu、FFH和scRNA是一个三元络合物，scRNA是一个结合的功能单位。这些结果表明，HBsu是枯草杆菌中能够与scRNA的Alu结构域结合的信号识别颗粒状颗粒的第三组分。在大肠杆菌中，4.5S RNA存在于蛋白质易位蛋白FFH(哺乳动物SRP54的细菌同源物)和蛋白质合成延伸因子G(EF-G)的复合体中。为了分析4.5S RNA在翻译中的功能，我们最初评估了4.5S RNA与核糖体在抗生素处理后与核糖体结合的敏感性，抗生素影响蛋白质合成的不同阶段。呋西地酸和维菌素使4.5S RNA与70年代核糖体部分共降解，表明4.5S RNA在核糖体转位和EF-G-GDP从核糖体释放之前进入核糖体。另一方面，4.5S RNA的耗尽导致了相当数量的EF-G保留在70S核糖体上。此外，4.5S RNA具有一个保守的十核苷酸序列(58GAAGCAGCCA67)，在23S RNA上的1068-1077位具有特征的EF-G结合位点。因此，我们通过凝胶迁移率改变分析来检查4.5S RNA的结构域IV区域的突变，包括这个保守的基序，是否干扰EF-G与23S RNA的结合。该基序中C62、G64或A67残基的任何突变都会取消竞争活性。因此，我们认为4.5S RNA与EF-G与核糖体的结合方式有关。此外，这一功能依赖于4.5S RNA的二级结构以及两个RNA之间保守的10个碱基序列。较少

项目成果

Nakamura K.: "Depletion of Escherichia coli 4.5S RNA leads to an increase in the amount of protein elongation factor EF-G associated with ribosomes"Eur. J. Biochem.. 259. 543-550 (1999)

Nakamura K.：“大肠杆菌 4.5S RNA 的消耗导致与核糖体相关的蛋白质延伸因子 EF-G 的量增加”Eur。

Yamane K.: "Signal tranduction, compretence development and protein secretion in Bacillus subtilis"Tanpakushitsu kakusan Koso. 44. 1467-1474 (1999)

Yamane K.：“枯草芽孢杆菌中的信号转导、功能发育和蛋白质分泌”Tanpakushitsu kakusan Koso。

Bunai K, Yamada K, Hayashi K, Nakamura K, Yamane K.: "Enhancing effect of Bacillus subtilis Ffh, a homologue of the SRP54 subunit of the mammalian signal recognition particle, on the binding of SecA to precursors of secretory proteins in vitro."J Biochem

Bunai K、Yamada K、Hayashi K、Nakamura K、Yamane K.：“枯草芽孢杆菌 Ffh（哺乳动物信号识别颗粒 SRP54 亚基的同源物）在体外增强 SecA 与分泌蛋白前体结合的作用。

Yamazaki T.: "Depletion of Bacillus subtilis histone-like protein, HBsu, causes defective protein translocation and induces upregulation of small cytoplasmic RNA"Biochem. Biophys. Res. Commun.. 258. 211-214 (1999)

Yamazaki T.：“枯草芽孢杆菌组蛋白样蛋白 HBsu 的消耗会导致有缺陷的蛋白易位并诱导小细胞质 RNA 的上调”Biochem。

Nakamura K.: "Bacillus subtilis histone-like protein, HBsu, is an integral component of a SRP-like particle that can bind the Alu domain of small cytoplasmic RNA"J. Biol. Chem.. 274. 13569-13576 (1999)

Nakamura K.：“枯草芽孢杆菌组蛋白样蛋白 HBsu 是 SRP 样颗粒的组成部分，可以结合小细胞质 RNA 的 Alu 结构域”J.