Active Disruption of an RNA-Protein Interaction by a DExH/D RNA Helicase
10.1126/science.291.5501.121
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

All aspects of cellular RNA metabolism and the replication of many viruses require DExH/D proteins that manipulate RNA in a manner that requires nucleoside triphosphates. Although DExH/D proteins have been shown to unwind purified RNA duplexes, most RNA molecules in the cellular environment are complexed with proteins. It has therefore been speculated that DExH/D proteins may also affect RNA-protein interactions. We demonstrate that the DExH protein NPH-II from vaccinia virus can displace the protein U1A from RNA in an active adenosine triphosphate–dependent fashion. NPH-II increases the rate of U1A dissociation by more than three orders of magnitude while retaining helicase processivity. This indicates that DExH/D proteins can effectively catalyze protein displacement from RNA and thereby participate in the structural reorganization of ribonucleoprotein assemblies.

Bibliography

Jankowsky, E., Gross, C. H., Shuman, S., & Pyle, A. M. (2001). Active Disruption of an RNA-Protein Interaction by a DExH/D RNA Helicase. Science, 291(5501), 121–125.

Authors 4
  1. Eckhard Jankowsky (first)
  2. Christian H. Gross (additional)
  3. Stewart Shuman (additional)
  4. Anna Marie Pyle (additional)
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  13. To unwind RNA NPH-II requires a single-strand overhang 3′ to the duplex region (23). Optimal helicase activity requires a 3′ overhang of at least 20 nucleotides (24). The affinity of NPH-II for blunt-end duplex RNA is low; no significant binding is observed at nanomolar concentrations of NPH-II (24).
  14. Unwinding of substrate in the absence of U1A can be described by a single exponential with a rate constant of k unwinding = 3.5 ± 0.4 min −1 which is in agreement with rate constants measured for unwinding of regular duplexes under these conditions (7). In the presence of NPH-II trap ∼70% of substrate was unwound; i.e. the overall processivity for unwinding this substrate is slightly lower then the overall processivity for unwinding a regular duplex with the same number of base pairs (bp) (7). U1A did not affect unwinding reactions with regular duplexes at the concentrations used (24).
  15. The RNA used for trapping dissociated U1A was based on the hairpin that forms the U1A binding site in the U1 small nuclear RNA (10). An RNA oligonucleotide of the sequence 5′-GGAGAACCAUUGCACUCCGGUUCUUC was prepared by chemical synthesis and purified as described (25).
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  17. Explicit equations describing the kinetic mechanism (Fig. 4D) were derived by considering the species I 1 as a fast intermediate such that dI 1 /dt = 0. The relative fractions (frac) of bound free and unwound substrate were described by frac[bound]=k2dk2+k2d·(1−e−k1t)+e−k1t frac[free]=k2k2+k2d·k1k3+k3d−k1·k3k3+k3d· (e−k1t−e−(k3+k3d)t)+k3dk3+k3d (1−e−k1t) frac[unwound]=k2k2+k2d·k3k3+k3d· 1−e−k1t−k1k3+k3d−k1·(e−k1t−e−(k3+k3d)t) where t is time. These equations were used to fit the normalized (26) time courses of reactions conducted in the presence of NPH-II trap RNA. Fitting was performed with Kaleidagraph (Synergy software). Values for k 2 /(k 2 + k 2d ) and for k 1 were obtained by fitting the time course of fraction[bound]. Values for k 3 and k 3d were computed by fitting fraction[free] and fraction[unwound] with fixed k 2 /k 2d and k 1 . The rate constants provided are average values calculated from three different time courses resulting in k 1 = 3.52 ± 0.15 min −1 k 2 /(k 2 + k 2d ) = 0.59 ± 0.02 k 3 = 1.04 ± 0.04 min −1 and k 3d = 0.40 ± 0.12 min −1 .
  18. The lower limit for k 2 was estimated by simulating the time course with the empirically determined rate constants but decreasing the values for k 2 . Noticable deviation from the observed time course was detected for values of k 2 < 50 min −1 ; i.e. the actual constant k 2 is necessarily larger than this value.
  19. For simulating the reaction without NPH-II trap rebinding of helicase to substrate was considered by adding three steps to the reaction scheme in Fig. 4D. (i) Fast binding of helicase to substrate-U1A complex: E + I′ 1 → ES where k 6 = 10 9 mol −1 · min −1 and the initial NPH-II concentration E 0 = 20 nM. (ii) Fast rebinding of helicase to substrate without U1A bound: E + I′ 2 → EI′ 2 where k 7 = 10 9 mol −1 · min −1 and the initial NPH-II concentration E 0 = 20 nM. (iii) Unwinding of rebound substrate without U1A bound: EI′ 2 → P where k 8 = 3.5 min −1 (14). Step (iii) represents multiple reactions. Simulations were performed with normalized (26) time courses using the KINSIM software package (27).
  20. This contrasts with the SNF2 family protein Mot1p which displaces the TATA box–binding protein from DNA in an ATP-dependent fashion (28) but lacks helicase activity.
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  24. E. Jankowsky et al. unpublished results.
  25. RNA oligonucleotides were prepared by chemical synthesis on an ABI 392 RNA/DNA synsthesizer using phosphoramidite chemistry (reagents were purchased from Glen Research Sterling VA). Crude oligonucleotides were deprotected according to standard protocols (29) and purified by denaturing PAGE. Duplexes were formed and purified as described previously (7).
  26. Amplitudes were corrected for the final reaction endpoint (normalized). Endpoints (t → ∞) were determined after 10 min of reaction without NPH-II trap. The endpoint values were determined to be as follows: frac[bound] obs (t → ∞) = 0.04 frac[free] obs - (t → ∞) = 0.02 and frac[unwound] obs (t → ∞) = 0.94. Amplitudes at a given t were corrected as follows: frac[bound](t) = (frac[bound] obs (t) − 0.04)/(1 − 0.04) frac[unwound](t) = frac[unwound] obs (t)/0.94 frac[free](t) = 1 − frac- [bound](t) − frac[unwound](t).
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  31. We thank K. Nagai for supplying purified U1A protein and for helpful discussion and S. Lomvardas for advice and assistance in visualizing RNA-protein complexes. Supported by grants from the NSF (NSF Young Investigator Award to A.M.P.) and the Matheson Foundation (A.M.P.). A.M.P. is an assistant investigator of the Howard Hughes Medical Institute.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:53 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 4:46 a.m.)
Indexed 1 month ago (Aug. 2, 2025, 12:23 a.m.)
Issued 24 years, 8 months ago (Jan. 5, 2001)
Published 24 years, 8 months ago (Jan. 5, 2001)
Published Print 24 years, 8 months ago (Jan. 5, 2001)
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@article{Jankowsky_2001, title={Active Disruption of an RNA-Protein Interaction by a DExH/D RNA Helicase}, volume={291}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.291.5501.121}, DOI={10.1126/science.291.5501.121}, number={5501}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Jankowsky, Eckhard and Gross, Christian H. and Shuman, Stewart and Pyle, Anna Marie}, year={2001}, month=jan, pages={121–125} }