RNA helicase activity probed at the single molecule level
Helicase activity will be observed only if CrhR unwinding is processive with the helicase remaining bound to the RNA substrate until strand separation is complete Helicase dissociation after a single unwinding step will not produce ssRNA, because the released helicase will be sequestered by the trap nucleic acid. The extent of unwinding obtained in the absence of trap is shown in lanes 2 and 3.
The trap effect was confirmed since the addition of trap nucleic acid prior to the dsRNA substrate abolished unwinding data not shown. Based on these results, CrhR unwinding proceeds through 25 bp of dsRNA without dissociating from the substrate. Helicase assays were conducted under single turnover conditions that do not allow reinitiation of unwinding once the helicase has dissociated from the initial RNA duplex. Standard helicase assays were performed with 80 ng A or ng B of recombinant CrhR for 10 and 40 min lanes 2 and 3.
Divalent cation and the indicated RNA trap 0. Unwinding, indicated by the appearance of the ssRNA product above background levels observed in the absence of protein lane 1 , indicates processivity of CrhR through the A or bp B duplex regions. In addition, the helicase and annealing reactions can occur concurrently to promote RNA strand exchange most probably through a branch migration mechanism.
This finding implies that these proteins utilize similar mechanisms to reconfigure nucleic acid secondary structure X-ray crystallographic analysis supports this proposal by revealing that these proteins possess similar three-dimensional conformations 1 , 2. The lack of RNA substrate specificity observed in vitro raises the question as to how the inappropriate resolution of RNA duplexes is prevented in vivo.
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One mechanism may involve a limit on helicase processivity, thereby preventing unwinding of more stable duplexes These values would not be expected to inhibit CrhR activity, because ATP levels do not vary significantly during light-dark transitions The observed specific activity of min —1 is at the upper range reported for helicases, similar to DbpA 28 and Ded1p CrhR also catalyzed bidirectional strand separation of RNA duplexes up to 25 bp in length but not a bp RNA duplex in the in vitro assays used here. Unwinding was also observed under conditions designed to prevent reassociation of CrhR once dissociation from the substrate RNA had occurred.
Taken together, these data suggested that CrhR is limited to a maximum of six to seven consecutive unwinding steps, assuming a step-size of 6 as reported for other RNA and DNA helicases 6 , Low helicase processivity may be a reflection of helicase removal from the macromolecular machine in which it functions in vivo or a reflection that its physiological function requires the unwinding of only a few base pairs as proposed for eIF-4A In addition, CrhR couples the unwinding and annealing activities to effect RNA rearrangements via strand exchange.
The reaction produces RNA duplexes whose structure is too stable to be resolved by helicase activity, an indication that annealing is more processive than unwinding. Processive annealing may be required in vivo , the stability of the products driving the reaction unidirectionally by preventing helicase activity. Although the mechanism by which CrhR promotes strand exchange remains to be elucidated, the reaction most probably proceeds through a branch migration process, CrhR initially interacting with and subsequently unwinding the duplex RNA while concurrently annealing a complementary RNA.
It has been proposed that p68 and 72 also promote RNA strand exchange through a branch migration complex, which represents an enzymatic function other than the isolated combination of the two reactions 8. A similar scenario occurs with CrhR in which strand exchange is more processive than unwinding. CrhR also preferentially initiates strand exchange from a dsRNA substrate, a further indication of a branch migration mechanism. It is possible that one or more of the activities observed in vitro is peripheral to its physiological role. Association with accessory factors may dictate which functions predominate in vivo as proposed for other helicases Although it is clear that the results obtained in vitro are produced by a combination of CrhR-catalyzed RNA unwinding and annealing, it is not entirely clear under which conditions one activity predominates.
The conditions that favor either reaction appear to depend on a complex interaction between all of the substrates with the dsRNA:ssRNA ratio being a crucial factor. Similar to CrhR, RNA-binding proteins have a narrow window of activity in which they switch from unwinding to annealing 3. Intracellular pH may also contribute to the predominating activity, as cyanobacterial cytoplasmic pH decreases from 7. Enhanced annealing at low pH in vitro may indicate that the annealing and unwinding reactions may predominate in the dark and light, respectively. This potential divergence of function may be reflected by the pattern of crhR expression, which is regulated by light-driven changes in the redox potential of the electron transport chain Although crhR transcript accumulates in the light, transcript and protein stability was unexpectedly enhanced in the dark, suggesting that CrhR activity is required under both conditions Whether CrhR unwinds in the light and anneals in the dark remains to be determined.
This implies that the active promotion of RNA structural rearrangements by CrhR coupling of helicase and annealing activities is required for cyanobacterial acclimation to a variety of environmental stresses. What is the fate of the ssRNA product of helicase activity? It is well known that the rearrangement of nucleic acid structure during a number of crucial cellular activities involves unwinding and annealing driven by independent proteins The ability of CrhR to perform both functions concurrently provides the ability to rapidly and efficiently convert one ribonucleoprotein structure into another.
We thank Dr. Nargang and members of the Owttrim laboratory for critical reading of the manuscript. Kujat-Choy and G. Owttrim, unpublished observation. The costs of publication of this article were defrayed in part by the payment of page charges. Section solely to indicate this fact. You'll be in good company. Journal of Lipid Research.
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Prev Next Table of Contents. This Article First Published on November 12, doi: Services Email this article to a friend Alert me when this article is cited Alert me if a correction is posted Alert me when eletters are published Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Download to citation manager Request Permissions. Responses Submit a Letter to the Editor. Google Scholar Articles by Chamot, D. Articles by Owttrim, G. Measurements were performed using MBP-tagged D1D2 to increase protein solubility under the experimental conditions.
The protein concentration was chosen so that all of the duplex substrate is bound in the open state at equilibrium Figure 5A. The fluorescence signal of the bound duplex substrate was quantified by using a Typhoon imager GE Healthcare, UK to measure the formation of a closed-state complex containing a single-stranded nucleic acid region, indicating duplex unwinding Figure 2—figure supplement 1.
In all cases, equilibrium was verified by additional assays for samples that were incubated for extended times up to approximately 4 hr , which gave the same unwinding profiles as those incubated for 1 hr. In these assays, a change in the fluorescence of the labeled duplex was seen upon unwinding and subsequent re-annealing to form a duplex with an unlabeled strand of the same sequence without a quencher present in excess Figure 2—figure supplement 2.
hillhurstcleaners.com/cli/alternate/cornerstones-of-health.php Annealing of these duplexes occurs within the dead time of mixing at the concentration of substrates used in these experiments. The apparent fraction of unwound duplex at various time points was quantified by using ImageJ and where appropriate fit to a first-order reaction to estimate an observed first-order rate constant k 1. The observed fluorescence anisotropy at increasing concentrations of protein was measured by using an EnVision Microplate Reader Perkin Elmer, Waltham, MA and was fit to a one-site binding model with a Hill coefficient to estimate the K d of single-stranded nucleic acid in the presence of increasing nucleotide.
Equilibrium was verified by carrying out assays on samples incubated for extended times up to 4 hr, which gave the same binding profiles as those incubated for 1 hr.
The fraction of bound duplex with increasing concentrations of MBP-tagged protein was quantified by using ImageJ and fit to a one-site binding model with a Hill coefficient to estimate a K d. In these cases, the fraction of free substrate was quantified and a K i was estimated from a one-site binding model. Binding of nucleotide and nucleic acid substrates to D1D2 was examined by size-exclusion chromatography. The formation of a closed-state complex in the presence of nucleic acid and NDP-BeF x therefore gives rise to a large change in A compared to A The absorbance and elution volume of the protein complexes above the background signal of the buffer were measured at and nm Table 1.
Related RNA helicases
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