Allosteric Activation of the ATPase Activity of the Escherichia coli RhlB RNA Helicase

Abstract

Helicase B (RhlB) is one of the five DEAD box RNA-dependent ATPases found in Escherichia coli. Unique among these enzymes, RhlB requires an interaction with the partner protein RNase E for appreciable ATPase and RNA unwinding activities. To explore the basis for this activating effect, we have generated a di-cistronic vector that overexpresses a complex comprising RhlB and its recognition site within RNase E, corresponding to residues 696 -762. Complex formation has been characterized by isothermal titration calorimetry, revealing an avid, enthalpyfavored interaction between the helicase and RNase E-(696 -762) with an equilibrium binding constant (K a ) of at least 1 ؋ 10 8 M ؊1 . We studied ATPase activity of mutants with substitutions within the ATP binding pocket of RhlB and on the putative interaction surface that mediates recognition of RNase E. For comparisons, corresponding mutations were prepared in two other E. coli DEAD box ATPases, RhlE and SrmB. Strikingly, substitutions at a phenylalanine near the Q-motif found in DEAD box proteins boosts the ATPase activity of RhlB in the absence of RNA, but completely inhibits it in its presence. The data support the proposal that the protein-protein and RNA-binding surfaces both communicate allosterically with the ATPase catalytic center. We conjecture that this communication may govern the mechanical power and efficiency of the helicases, and is tuned in individual helicases in accordance with cellular function.RNA helicases are a diverse set of proteins found in all three kingdoms of life that possess the ability to unwind short stretches of RNA duplexes in reactions that require the hydrolysis of nucleoside triphosphates (1). They are the largest group of enzymes in eukaryotic RNA metabolism and are involved in virtually all aspects of cellular RNA manipulation including transcription, splicing, RNA nuclear export, and ribosome biogenesis (2, 3). The DEAD box helicases are grouped in helicase superfamily 2, whose members contain nine conserved motifs, including the Walker B motif DEAD from which they take their common name (Fig. 1). Escherichia coli contains five genes encoding for DEAD box proteins; csdA (formerly called deaD), dbpA, rhlB, rhlE, and srmB (4). The gene products have a common core of 350 -400 amino acids, consisting of two domains that have the same fold found in the DNA-recombination protein RecA. Flanking this two-domain core are variable regions that are thought to be responsible for the differing properties and functions of the five helicases (4).DEAD box helicases can unwind short RNA duplexes that are no more than two helical turns in length (ϳ22 bp), and some helicases can displace avidly bound proteins from RNA (1, 5). A role of DEAD box RNA helicases as nonspecific RNA unfolding enzymes has been described (6). Yang and Jankowsky (7) have elegantly shown that DEAD box helicases use a single strand RNA region to facilitate loading onto duplex RNA where only a few base pairs are disrupted in an ATP-dependent manner, leading to destabiliza...

The regulatory protein RraA modulates RNA-binding and helicase activities of the E. coli RNA degradosome

“…RraA forms a stable ternary complex with RNase E and helicase RhlB, in which all components can interact RhlB helicase binds strongly to the isolated 69-residue segment of RNase E that lies between the RBD and AR2 sites (K D of z10 nM) (Worrall et al 2008b). We tested whether RraA would bind to the stable binary complex of RhlB and RNase E(628-843), which encompasses the 69-residue helicase binding site and the flanking RNA-binding regions that were shown to be required for RraA binding in the previous section (Fig.…”

“…RNase E(628-843) construct is a fusion of RNase E sequence with a 13-residue leader and 20-residue C-terminal tail derived from its expression vector, pET11c. RNase E(696-762)/RhlB and RNase E(696-762)/RhlB(1-397) were purified as described previously (Worrall et al 2008b). During purification of these constructs, fractions from chelating chromatography lacking RNase E(696-762) were used as the source of RhlB or RhlB(1-397) alone.…”

“…As RhlB has poor ATPase activity in the absence of RNase E and RNA (Worrall et al 2008b), we used for the assay the avid complex of RhlB with its 69-residue recognition segment of RNase E that lacks the RNA-binding segments ]. The ATPase activity of the RhlB/RNase E(696-762) complex was stimulated slightly by RraA in the absence of RNA substrate (see the table in Fig.…”

The Q motif of a viral packaging motor governs its force generation and communicates ATP recognition to DNA interaction

“…In previous work examining the Q motif of Rh1B helicase, Worrall et al conjectured that the communication between the ATPase catalytic center and the RNA-binding surface through the Q motif governs the mechanical power and efficiency of RNA helicases and is tuned to the cellular function of the helicase (15). Also, in the crystal structure of T4's gp17 (N360-ED), it was discovered that a structural difference occurred between the apo and ADP/ATP-bound states in the adenineinteracting loop (Cys-125-Arg-140) near Q143, suggesting that T4's Q motif (shown in Fig.…”

“…It was further proposed that the Q motif functions as a molecular on-off switch for ATP hydrolysis and helicase activity (14). Mutation studies in the Q motif in RNA helicases concluded that, at minimum, it directly regulates the affinity of the protein for the substrate (RNA) through conformational changes associated with nucleotide binding (14,15). Also, through sequence alignments of various terminases, Mitchell and Rao suggested that bacteriophage may have a deviant Walker A motif (76KSARVGYS83 unlike the classic Walker GXXXXGKS/T) responsible for interactions with the ␤ and ␥ phosphates of ATP (10,16,17).…”