Potential regulatory interactions of Escherichia coli RraA protein with DEAD-box helicases.

Members of the DEAD-box family of RNA helicases contribute to virtually every aspect of RNA metabolism, in organisms from all domains of life. Many of these helicases are constituents of multicomponent assemblies, and their interactions with partner proteins within the complexes underpin their activities and biological function. In Escherichia coli the DEAD-box helicase RhlB is a component of the multienzyme RNA degradosome assembly, and its interaction with the core ribonuclease RNase E boosts the ATP-dependent activity of the helicase. Earlier studies have identified the regulator of ribonuclease activity A (RraA) as a potential interaction partner of both RNase E and RhlB. We present structural and biochemical evidence showing how RraA can bind to, and modulate the activity of RhlB and another E. coli DEAD-box enzyme, SrmB. Crystallographic structures are presented of RraA in complex with a portion of the natively unstructured C-terminal tail of RhlB at 2.8-Å resolution, and in complex with the C-terminal RecA-like domain of SrmB at 2.9 Å. The models suggest two distinct mechanisms by which RraA might modulate the activity of these and potentially other helicases.

SrmB Rescues Trapped Ribosome Assembly Intermediates.

RNA helicases play various roles in ribosome biogenesis depending on the ribosome assembly pathway and stress state of the cell. However, it is unclear how most RNA helicases interact with ribosome assembly intermediates or participate in other cell processes to regulate ribosome assembly. SrmB is a DEAD-box helicase that acts early in the ribosome assembly process, although very little is known about its mechanism of action. Here, we use a combined quantitative mass spectrometry/cryo-electron microscopy approach to detail the protein inventory, rRNA modification state, and structures of 40S ribosomal intermediates that form upon SrmB deletion. We show that the binding site of SrmB is unperturbed by SrmB deletion, but the peptidyl transferase center, the uL7/12 stalk, and 30S contact sites all show severe assembly defects. Taking into account existing data on SrmB function and the experiments presented here, we propose several mechanisms by which SrmB could guide assembling particles from kinetic traps to competent subunits during the 50S ribosome assembly process.