Structural Requirement in Clostridium perfringens Collagenase mRNA 5′ Leader Sequence for Translational Induction through Small RNA-mRNA Base Pairing

The Gram-positive anaerobic bacteriumClostridium perfringens is pathogenic to humans and animals, and the production of its toxins is strictly regulated during the exponential phase. We recently found that the 5′ leader sequence of thecolA transcript encoding collagenase, which is a major toxin of this organism, is processed and stabilized in the presence of the small RNA VR-RNA. The primarycolA 5′-untranslated region (5′UTR) forms a long stem-loop structure containing an internal bulge and masks its own ribosomal binding site. Here we found that VR-RNA directly regulatescolA expression through base pairing withcolA mRNAin vivo . However, when the internal bulge structure was closed by point mutations incolA mRNA, translation ceased despite the presence of VR-RNA. In addition, a mutation disrupting thecolA stem-loop structure induced mRNA processing and ColA-FLAG translational activation in the absence of VR-RNA, indicating that the stem-loop and internal bulge structure of thecolA 5′ leader sequence is important for regulation by VR-RNA. On the other hand, processing was required for maximal ColA expression but was not essential for VR-RNA-dependentcolA regulation. Finally,colA processing and translational activation were induced at a high temperature without VR-RNA. These results suggest that inhibition of thecolA 5′ leader structure through base pairing is the primary role of VR-RNA incolA regulation and that thecolA 5′ leader structure is a possible thermosensor.