Item

Abstract

Many bacteria harbor RNA-dependent nucleoside-triphosphatases of the DEAH/RHA family, whose molecular mechanisms and cellular functions are poorly understood. 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, crys-
tal 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 in combination with cross-linking/mass spectrometry revealed ratchet-like domain movements upon RNA engagement, much more pronounced
than hitherto observed in related eukaryotic DEAH/RHA enzymes. Structure-based functional analyses delineated transient interdomain contact sites that support substrate loading and unwinding, suggest-
ing that similar conformational changes support RNA translocation. Consistently, modeling studies showed that analogous dynamic intramolecular contacts are not possible in the related but helicase-inactive
RNA-dependent nucleoside-triphosphatase, HrpB. Our results indicate that HrpA may be an interesting target to interfere with bacterial
tolerance toward certain antibiotics and suggest possible interfering strategies.