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Functional analysis of eubacterial small cytoplasmic RNA (scRNA)

真细菌细胞质小 RNA (scRNA) 的功能分析

基本信息

批准号：
07660091
负责人：
NAKAMURA Kouji
金额：
$ 0.7万
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：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

B.subtilis scRNA is a member of SRP RNA family. By depleting scRNA in vivo, we have obtained a direct evidence that scRNA is essential for viability, protein secretion and translation. It is transcribed as a 354-nt primary transcript and processed at both 5' and 3' sites. In precursor, 5' and 3' flanking sequence encompassing the processing sites is complement. Therefore, it is predicted that both processing sites located within a double stranded stem structure. This substrate specificity is identical to that of well konwn ribonuclease III (RNaseIII). To examine the function of Bs-RNaseIII in processing of pre-scRNA,in vitro ^<32>P-labeled pre-scRNA (15ng) was incubated with the purified Bs-RNaseIII (1.5ng). Results indicate that pre-scRNA is primarily cleaved by Bs-RNaseIII at both 5' and 3' sites.Based on human SRP RNA,all most eukaryotic SRP RNA can be divided into four functional domains (domains I to IV). In addition to domain III,domains I and II are absent in most eubacteria except B.subtilis. The SRP RNA (scRNA) of B.subtilis consists of domains I,II and IV.Functional analysis demonstrated that deletion of domains I and/or II blocked formation of heat-resistant spore. Moreover, SRP RNA of another spore-forming bacteria, clostridium perfringens, contains three domains, and can restore the growth and sporulation of B.subtilis conditional mutant, suggesting that both domains I and II are required for sporulation. However, more extended phylogenetic studies revealed that non-spore forming gram-positive bacteria, such as staphylococcus aureus, Streptococcus pyogenesis, Corynebacterium glutamicum and Listeria monocytogenesis, also contain domains I and II like B.subtilis. The domains I and II are conserved throughout the evolution of gram-positive bacteria. This fact indicates that other important function will be included in domains I and II.

枯草杆菌单链RNA是SRP RNA家族的成员。通过在体内耗尽scRNA，我们已经获得了一个直接的证据，表明scRNA对于生存、蛋白质分泌和翻译是必不可少的。它被转录为354-nT的初级转录本，并在5‘和3’端加工。在前体中，包含加工位点的5‘和3’侧翼序列是互补的。因此，可以预测，这两个加工位点都位于双链茎结构内。这种底物特异性与广为人知的核糖核酸酶III(RNaseIII)相同。为了研究Bs-RNaseIII在前-scRNA加工过程中的作用，将纯化的Bs-RNaseIII(1.5 ng)与P标记的Pre-scRNA(15 Ng)体外孵育。结果表明，前-scRNA主要被BS-RNaseIII在5‘和3’端切割，根据人类SRP RNA，所有的真核SRP RNA都可以分为四个功能结构域(结构域I到IV)。除了结构域III，除枯草杆菌外，大多数真细菌中都不存在结构域I和结构域II。枯草杆菌SRP RNA(ScRNA)由结构域I、II和IV组成。功能分析表明，结构域I和/或II的缺失阻碍了耐热孢子的形成。另外，产气荚膜梭菌的SRP RNA含有3个结构域，能够恢复枯草杆菌条件突变株的生长和产孢量，表明I和II结构域都是产孢所必需的。然而，更广泛的系统发育研究发现，非芽胞形成革兰氏阳性菌，如金黄色葡萄球菌、化脓性链球菌、谷氨酸棒杆菌和单核细胞增多性李斯特菌，也含有枯草杆菌等结构域I和II。结构域I和II在革兰氏阳性细菌的进化过程中是保守的。这一事实表明，其他重要职能将包括在领域I和II中。