Functional analysis of eubacterial small cytoplasmic RNA (scRNA)

枯草芽孢杆菌scRNA是SRP RNA家族的一员。通过在体内消耗scRNA，我们获得了scRNA对生存能力、蛋白质分泌和翻译至关重要的直接证据。它转录为354-nt的主要转录本，并在5‘和3’位点进行加工。在前体中，包含加工位点的5‘和3’侧翼序列是补体。因此，预测这两个加工位点位于双链茎结构内。这种底物特异性与众所周知的核糖核酸酶III （RNaseIII）相同。为了检测Bs-RNaseIII在pre-scRNA加工中的功能，将体外^<32> p标记的pre-scRNA （15ng）与纯化的Bs-RNaseIII （1.5ng）孵育。结果表明，pre-scRNA主要被Bs-RNaseIII在5‘和3’位点切割。基于人类SRP RNA，大多数真核生物的SRP RNA可分为四个功能域（结构域I至结构域IV）。除枯草芽孢杆菌外，大多数真细菌中不存在结构域III和结构域II。枯草芽孢杆菌SRP RNA （scRNA）由I、II和iv结构域组成，功能分析表明，I和/或II结构域的缺失阻断了耐热孢子的形成。此外，另一种产孢细菌产气荚膜梭菌的SRP RNA包含三个结构域，可以恢复枯草芽孢杆菌条件突变体的生长和产孢，表明产孢需要两个结构域I和II。然而，更广泛的系统发育研究表明，非孢子形成的革兰氏阳性细菌，如金黄色葡萄球菌、化脓性链球菌、谷氨酸棒状杆菌和单核增生李斯特菌，也像枯草芽孢杆菌一样含有结构域I和II。结构域I和II在革兰氏阳性菌的进化过程中是保守的。这一事实表明，其他重要功能将包括在领域一和领域二。

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.