Functional analysis of eubacterial small cytoplasmic RNA (scRNA)

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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.