Functional Analysis of Vibrio vulnificus Orthologs of Escherichia coli RraA and RNase E

Abstract

RNase E has a pivotal role in the degradation and processing of RNAs in Escherichia coli, and protein inhibitors RraA and RraB control its enzymatic activity. The halophilic patho-genic bacterium Vibrio vulnificus also expresses orthologs of RNase E and RraA-RNase EV, RraAV1, and RraAV2 (herein renamed as VvRNase E, VvRraA1, and VvRraA2). A previous study showed that VvRraA1 actively inhibits the ribonucleolytic activity of VvRNase E by interacting with the C-terminal region of VvRNase E. However, the molecular mechanism underlying the effect of VvRraA1 on the ribonucleolytic activity of VvRNase E has not yet been elucidated. In this study, we report that the oligomer formation of VvRraA proteins affects binding efficiency to VvRNase E as well as inhibitory activity on VvRNase E action. The hexameric structure of VvRraA1 was converted to lower oligomeric forms when the Cys 9 residue was substituted with an Asp residue (VvRraA1-C9D), showing decreased inhibi-tory activity of VvRraA1 on VvRNase E in vivo. These results indicated that the intermolecu-lar disulfide linkage contributed critically to the hexamerization of VvRraA1 for its proper function. On the contrary, the VvRraA2 that existed in a trimeric state did not bind to or inhibit VvRNase E. An in vitro cleavage assay further showed the reduced inhibitory effect of VvRraA-C9D on VvRNase E activity compared to wild-type VvRraA1. These findings provide insight into how VvRraA proteins can regulate VvRNase E action on its substrate RNA in V. vulnificus. In addition, based on structural and functional comparison of RraA homo-logs, we suggest that hexameric assembly of RraA homologs may well be required for their action on RNase E-like proteins.

Functional implications of hexameric assembly of RraA proteins from Vibrio vulnificus

“…To test whether structural changes in VvRraA1 protein affect its known inhibitory effect on ribonucleolytic activity of VvRNase E (VvRne) [ 6 ], and whether VvRraA2 and VvRraA2-C9D can modulate the activity of VvRne, we investigated in vivo activity of these VvRraA proteins. We first assessed the growth rate of DK001 cells co-expressing VvRraA1, VvRraA2, or C9D mutants.…”

“…We first assessed the growth rate of DK001 cells co-expressing VvRraA1, VvRraA2, or C9D mutants. DK001 contains a deletion in the chromosomal rne gene that is complemented by plasmid-borne VvRne (pLAC-RNEV2), which expresses a full-length VvRne with a hexahistidine tag at the C-terminus under the control of an IPTG-inducible lacUV5 promoter [ 6 ]. To co-express the VvRraA proteins and their C9D mutants, a compatible Km r plasmid that expresses VvRraA1, VvRraA1-C9D, VvRraA2, and VvRraA2-C9D with hexahistidine tags at their C-termini under the control of an arabinose-inducible promoter (pKAN6B-VvRraA1, pKAN6B-VvRraA2, pKAN6B-VvRraA1-C9D, or pKAN6B-VvRraA2-C9D, respectively) was introduced into DK001 cells.…”

“…The construction of an E . coli strain that contains a deletion in the rne gene and expresses full-length VvRraA1 (DK001) has been described previously [ 6 ]. The E .…”

“…The depletion, adventitious overexpression, or increased enzymatic activity of RNase E causes growth retardation of cells [ 5 – 7 ]. Thus, the concentration of RNase E is maintained relatively stable in vivo through the autoregulatory mechanism such that the enzyme cleaves the 5′-untranslated region of its own mRNA when its activity exceeds cellular needs [ 6 , 8 ]. The endonucleolytic activity of RNase E is controlled by protein inhibitors RraA and RraB (regulator of ribonuclease activity A or B).…”

“…coli RNase E and two RraA-like proteins, herein renamed as VvRNase E, VvRraA1, and VvRraA2. The primary amino acid sequence of VvRNase E reveals 86.4% similarity with RNase E, and VvRraA1 and VvRraA2 have 80.1% and 59% amino acid sequence similarity with RraA, respectively [ 6 ]. Recent studies showed that VvRNase E has conserved enzymatic properties and VvRraA1efficiently inhibits the activity of both RNase E and VvRNase E [ 6 , 18 , 19 ].…”