Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor

Most nuclear receptors (NRs) bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs) involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR) belongs to the steroid hormone nuclear receptor (SHR) family and shares strong similarity in its DNA-binding domain (DBD) with that of the estrogen receptor (ER). In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3), but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs)], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS), non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3), such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3), where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half-site is followed by a weaker one with degenerated sequence

Exploring TAR–RNA aptamer loop–loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance

In HIV-1, trans-activation of transcription of the viral genome is regulated by an imperfect hairpin, the trans-activating responsive (TAR) RNA element, located at the 5′ untranslated end of all viral transcripts. TAR acts as a binding site for viral and cellular proteins. In an attempt to identify RNA ligands that would interfere with the virus life-cycle by interacting with TAR, an in vitro selection was previously carried out. RNA hairpins that formed kissing-loop dimers with TAR were selected [Ducongé F. and Toulmé JJ (1999) RNA, 5:1605–1614]. We describe here the crystal structure of TAR bound to a high-affinity RNA aptamer. The two hairpins form a kissing complex and interact through six Watson–Crick base pairs. The complex adopts an overall conformation with an inter-helix angle of 28.1°, thus contrasting with previously reported solution and modelling studies. Structural analysis reveals that inter-backbone hydrogen bonds between ribose 2′ hydroxyl and phosphate oxygens at the stem-loop junctions can be formed. Thermal denaturation and surface plasmon resonance experiments with chemically modified 2′-O-methyl incorporated into both hairpins at key positions, clearly demonstrate the involvement of this intermolecular network of hydrogen bonds in complex stability

Nucleic Acids Res

Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5'-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies

NMR structure of a kissing complex formed between the TAR RNA element of HIV-1 and a LNA-modified aptamer

The trans-activating responsive (TAR) RNA element located in the 5′ untranslated region of the HIV-1 genome is a 57-nt imperfect stem-loop essential for the viral replication. TAR regulates transcription by interacting with both viral and cellular proteins. RNA hairpin aptamers specific for TAR were previously identified by in vitro selection [Ducongé,F. and Toulmé,J.J. (1999) In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1. RNA, 5, 1605–1614]. These aptamers display a 5′-GUCCCAGA-3′ consensus apical loop, partially complementary to the TAR one, leading to the formation of a TAR–aptamer kissing complex. The conserved GA combination (underlined in the consensus sequence) has been shown to be crucial for the formation of a highly stable complex. To improve the nuclease resistance of the aptamer and to increase its affinity for TAR, locked nucleic acid (LNA) nucleotides were introduced in the aptamer apical loop. LNA are nucleic acids analogues that contain a 2′-O,4′-C methylene linkage and that raise the thermostablity of duplexes. We solved the NMR solution structure of the TAR–LNA-modified aptamer kissing complex. Structural analysis revealed the formation of a non-canonical G•A pair leading to increased stacking at the stem-loop junction. Our data also showed that the introduction of LNA residues provides an enhanced stability while maintaining a normal Watson–Crick base pairing with a loop–loop conformation close to an A-type

HEXIM1 targets a repeated GAUC motif in the riboregulator of transcription 7SK and promotes base pair rearrangements

7SK snRNA, an abundant RNA discovered in human nucleus, regulates transcription by RNA polymerase II (RNAPII). It sequesters and inhibits the transcription elongation factor P-TEFb which, by phosphorylation of RNAPII, switches transcription from initiation to processive elongation and relieves pauses of transcription. This regulation process depends on the association between 7SK and a HEXIM protein, neither isolated partner being able to inhibit P-TEFb alone. In this work, we used a combined NMR and biochemical approach to determine 7SK and HEXIM1 elements that define their binding properties. Our results demonstrate that a repeated GAUC motif located in the upper part of a hairpin on the 5′-end of 7SK is essential for specific HEXIM1 recognition. Binding of a peptide comprising the HEXIM Arginine Rich Motif (ARM) induces an opening of the GAUC motif and stabilization of an internal loop. A conserved proline-serine sequence in the middle of the ARM is shown to be essential for the binding specificity and the conformational change of the RNA. This work provides evidences for a recognition mechanism involving a first event of induced fit, suggesting that 7SK plasticity is involved in the transcription regulation

Determination of refractive index increment ratios for protein-nucleic acid complexes by surface plasmon resonance.

International audienceThree nucleic acid-protein complexes of 1:1 stoichiometry were analyzed by surface plasmon resonance on a Biacore biosensor to test whether or not proteins and nucleic acids yielded similar refractive index increments on binding. The expected maximum response in resonance units, (RU(exp))(max), and the observed one, (RU(obs))(max), on saturation of immobilized targets by interacting partners were compared to determine the ratio of (deltan/deltaC)(protein) to (deltan/deltaC)(nucleic acid), where n is the refractive index at the surface and C is the concentration of one partner. Our results suggest that proteins and nucleic acids behave similarly and that the discrepancy between the expected and observed maximum responses for such complexes reflects inaccurate evaluation of the binding responses. Therefore, no correction of the instrument response is required for protein and nucleic acid interaction studies on a Biacore biosensor

Application of NMR Spectroscopy to Determine the 3D Structure of Small Non-Coding RNAs

Many RNA architectures were discovered to be involved in a wide range of essential biological processes in all organisms from carrying genetic information to gene expression regulation. The remarkable ability of RNAs to adopt various architectures depending on their environment enables the achievement of their myriads of biological functions. Nuclear Magnetic Resonance (NMR) is a powerful technique to investigate both their structure and dynamics. NMR is also a key tool for studying interactions between RNAs and their numerous partners such as small molecules, ions, proteins, or other nucleic acids. In this chapter, to illustrate the use of NMR for 3D structure determination of small noncoding RNA, we describe detailed methods that we used for the yeast C/D box small nucleolar RNA U14 from sample preparation to 3D structure calculation

Enhancement of water suppression by radiation damping-based manipulation of residual water in Jump and Return NMR experiments

International audienceIt is shown in this Letter that amplification of radiation damping obtained by electronic feedback between consecutive transients of a Jump-Return (JR) experiment, residual in-plans water magnetization is suppressed, leading to substantial enhancement of water suppression. Application of this approach to RNA spectroscopy, where JR is particularly useful, is shown. (C) 2001 Elsevier Science B.V. All rights reserved