Crystal structure of Streptomyces coelicolor RraAS2, an unusual member of the RNase E inhibitor RraA protein family.

Bacterial ribonuclease E (RNase E) plays a crucial role in the processing and decay of RNAs. A small protein named RraA negatively regulates the activity of RNase E via protein-protein interaction in various bacteria. Recently, RraAS1 and RraAS2, which are functional homologs of RraA from Escherichia coli, were identified in the Gram-positive species Streptomyces coelicolor. RraAS1 and RraAS2 inhibit RNase ES ribonuclease activity in S. coelicolor. RraAS1 and RraAS2 have a C-terminal extension region unlike typical bacterial RraA proteins. In this study, we present the crystal structure of RraAS2, exhibiting a hexamer arranged in a dimer of trimers, consistent with size exclusion chromatographic results. Importantly, the C-terminal extension region formed a long α-helix at the junction of the neighboring subunit, which is similar to the trimeric RraA orthologs from Saccharomyces cerevisiae. Truncation of the C-terminal extension region resulted in loss of RNase ES inhibition, demonstrating its crucial role. Our findings present the first bacterial RraA that has a hexameric assembly with a C-terminal extension α-helical region, which plays an essential role in the regulation of RNase ES activity in S. coelicolor.

RraAS1 inhibits the ribonucleolytic activity of RNase ES by interacting with its catalytic domain in Streptomyces coelicolor.

RraA is a protein inhibitor of RNase E, which degrades and processes numerous RNAs in Escherichia coli. Streptomyces coelicolor also contains homologs of RNase E and RraA, RNase ES and RraAS1/RraAS2, respectively. Here, we report that, unlike other RraA homologs, RraAS1 directly interacts with the catalytic domain of RNase ES to exert its inhibitory effect. We further show that rraAS1 gene deletion in S. coelicolor results in a higher growth rate and increased production of actinorhodin and undecylprodigiosin, compared with the wild-type strain, suggesting that RraAS1-mediated regulation of RNase ES activity contributes to modulating the cellular physiology of S. coelicolor.

RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity.

RraA is a protein inhibitor of RNase E (Rne), which catalyzes the endoribonucleolytic cleavage of a large proportion of RNAs in Escherichia coli. The antibiotic-producing bacterium Streptomyces coelicolor also contains homologs of RNase E and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2, respectively. Here, we report that RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity. Analyses of the steady-state level of RNase E substrates indicated that coexpression of RraAS2 in E. coli cells overproducing Rns effectively inhibits the ribonucleolytic activity of full-length RNase ES, but its inhibitory effects were moderate or undetectable on other truncated forms of Rns, in which the N- or/and C-terminal scaffold domain was deleted. In addition, RraAS2 more efficiently inhibited the in vitro ribonucleolytic activity of RNase ES than that of a truncated form containing the catalytic domain only. Coimmunoprecipitation and in vivo cross-linking experiments further showed necessity of both scaffold domains of RNase ES for high-affinity binding of RraAS2 to the enzyme, resulting in decreased RNA-binding capacity of RNase ES. Our results indicate that RraAS2 is a protein inhibitor of RNase ES and provide clues to how this inhibitor affects the ribonucleolytic activity of RNase ES.