Many bacteria harbor RNA-dependent nucleoside-triphosphatases of the DEAH/RHA family (1), whose molecular mechanisms and cellular functions are poorly understood (2). Here, we show that the Escherichia coli DEAH/RHA protein, HrpA, is an ATP-dependent 3’-to-5’ RNA helicase, and that the RNA helicase activity of HrpA influences bacterial survival under antibiotics treatment. Limited proteolysis, crystal structure analysis and functional assays showed that HrpA contains an N-terminal DEAH/RHA helicase cassette preceded by a unique N-terminal domain and followed by a large C-terminal region that modulates the helicase activity. Structures of an expanded HrpA helicase cassette in the apo and RNA-bound states revealed ratchet-like domain movements upon RNA engagement much more pronounced than hitherto observed in related eukaryotic DEAH/RHA enzymes (Fig. 1). Consistent with similar conformational changes supporting RNA translocation, structure-based functional analyses delineated transient inter-domain contact sites that support substrate loading and unwinding. Analogous dynamic intramolecular contacts are not possible in the related, but helicase-inactive, RNA-dependent nucleoside-triphosphatase, HrpB (3,4). Our results indicate that HrpA may be an interesting target to interfere with bacterial tolerance toward certain antibiotics and suggest possible interfering strategies.
Fig. 1. Top, Domain composition of the E. coli DEAH/RHA-type RNA helicase, HrpA. NTD, N-terminal domain; RecA1/2, RecA-like domains; WH, winged-helix domain; HB, helical bundle (“ratchet”) domain; OB, oligonucleotide/oligosaccharide-binding domain; CON, connector domain; CTR, C-terminal region. Bottom, comparison of the crystal structures of the expanded helicase cassette of E. coli HrpA in the apo (left) and RNA-bound (right) states.
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