Abstract
Helicase B (RhlB) is one of the five DEAD box RNA-depend-ent 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, enthalpy-favored interaction between the helicase and RNase E-(696–762) with an equilibrium binding constant (Ka) of at least 1 × 108 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.
Highlights
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]
We observe that the RNase E/RhlB
We examined the ATPase activity of the Y383A variant, but no detectable activity was observed in the absence or presence of RNase E-(696 –762) or RNA, which may be due to the oligomerization of this mutant
Summary
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]. The crystal structure of the Drosophila DEAD box helicase Vasa in complex with a non-hydrolyzable ATP analogue and single-stranded RNA lends support to the proposed mechanism of duplex unwinding by the DEAD box family [8] This complex structure shows that ATP binding promotes relative movement of the two RecA-like domains to induce a closed form of the helicase. Turning to examples of bacterial helicase complexes, the E. coli DEAD box ATPase RhlB forms part of the multiprotein assembly known as the RNA degradosome. This large complex comprises the endoribonuclease, RNase E, the exoribonuclease, polynucleotide phosphorylase, and the glycolytic enzyme, enolase. We observe that the RNase E/RhlB interaction decreases the RNA affinity of the helicase.
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