Structural transitions in the RNA 7SK 5' hairpin and their effect on HEXIM binding.

7SK RNA, as part of the 7SK ribonucleoprotein complex, is crucial to the regulation of transcription by RNA-polymerase II, via its interaction with the positive transcription elongation factor P-TEFb. The interaction is induced by binding of the protein HEXIM to the 5' hairpin (HP1) of 7SK RNA. Four distinct structural models have been obtained experimentally for HP1. Here, we employ computational methods to investigate the relative stability of these structures, transitions between them, and the effects of mutations on the observed structural ensembles. We further analyse the results with respect to mutational binding assays, and hypothesize a mechanism for HEXIM binding. Our results indicate that the dominant structure in the wild type exhibits a triplet involving the unpaired nucleotide U40 and the base pair A43-U66 in the GAUC/GAUC repeat. This conformation leads to an open major groove with enough potential binding sites for peptide recognition. Sequence mutations of the RNA change the relative stability of the different structural ensembles. Binding affinity is consequently lost if these changes alter the dominant structure

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

Approche structurale du rôle de l ARN 7SK comme régulateur de la transcription eucaryote

7SK est un ARN non-codant présent dans le noyau des métazoaires. Avec la protéine HEXIM1, 7SK séquestre et inhibe l'activité kinase du facteur d'élongation de transcription, P-TEFb. P-TEFb stimule l'élongation productive de l'ARN polymérase II. L'association de 7SK à différents partenaires protéiques peut jouer un rôle clé dans la régulation de la transcription. Nous avons identifié et caractérisé les déterminants structurels de 7SK pour son interaction avec ses partenaires protéiques, et en particulier avec HEXIM1. Des études SHAPE et SAXS ont suggéré que 7SK est une molécule semi-compact, flexible et modulaire. Trois sous-domaines structurels autonomes ont été identifiés. L'un d'eux, HP1 (nucléotides 24 à 87), interagit avec HEXIM1. Une cartographie par RMN a montré que l ARM de HEXIM1 est capable de lier spécifiquement le motif GAUC conservé dans la région apicale de HP1. Les Us extrudées qui bordent ce motif ont un rôle essentiel dans la reconnaissance. Lors de la liaison, la tige comprenant le motif GAUC s'ouvre et la paire de base A39/G68 est formée. Nous avons constaté par des expériences EMSAs que des mutations du motif GAUC, des Us extrudées ou de la boucle interne dans la région centrale de HP1, nuisent à la liaison à HEXIM1, alors que les mutations dans la région basale de la tige n'affectent pas l'interaction. Des expériences MS ont montré que HEXIM1 lie HP1 sous forme de dimère. En utilisant une HEXIM monomère, nous avons constaté que deux monomères interagissent avec HP1. Ces résultats confirment l'existence d'un deuxième site de liaison de HP1, dont l'emplacement précis reste encore à déterminer.7SK is a non-coding RNA present in the nucleus of metazoans. Together with the HEXIM1 protein, 7SK sequesters and inhibits the kinase activity of the Positive Transcription Elongation Factor, P-TEFb. P-TEFb stimulates the productive elongation by releasing the RNA polymerase II from the promoter-proximal pausing. The association of 7SK to different protein partners may play a key role in the regulation of the RNAPII transcription. We identified and characterized the structural determinants in 7SK for its interaction with its protein partners, and in particular with HEXIM1. SHAPE and SAXS studies suggested that 7SK is a semi-compact, flexible and modular molecule. Three structural, autonomous subdomains were identified. One of them, HP1 (nucleotides 24 to 87), binds to HEXIM1. NMR mapping showed that the ARM of HEXIM1 is able to specifically bind the conserved GAUC repeated motif in the apical region of HP1. The bulged Us encompassing this motif have an essential role in the recognition. Upon the binding, the GAUC motif stem opens and the base pair A39/G68 is formed. Using EMSA, we found that mutations of the GAUC motif, of the bulged Us or of the internal loop in the middle region of HP1, highly impair the binding to HEXIM1, whereas mutations in the basal stem region do not affect the interaction. Our investigation by MS showed that the HEXIM1 binds preferentially HP1 as a dimer. Using a monomer HEXIM, we found that two monomers interact with HP1. These results support the existence of a second binding site in HP1, close to the GAUC motif. Further work needs to be done to establish the precise location of this second binding site.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

The La-related proteins: Structures and interactions of a versatile superfamily of RNA binding proteins

International audienc

Aminoacylation at the atomic level in class IIa aminoacyl-tRNA synthetases

The crystal structures of histidyl- (HisRS) and threonyl-tRNA synthetase (ThrRS) from E. coli and glycyl-tRNA synthetase (GlyRS) from T. thermophilus, all homodimeric class IIa enzymes, were determined in enzyme-substrate and enzyme-product states corresponding to the two steps of aminoacylation. HisRS was complexed with the histidine analog histidinol plus ATP and with histidyl-adenylate, while GlyRS was complexed with ATP and with glycyl-adenylate; these complexes represent the enzyme-substrate and enzyme-product states of the first step of aminoacylation, i.e. the amino acid activation. In both enzymes the ligands occupy the substrate-binding pocket of the N-terminal active site domain, which contains the classical class II aminoacyl-tRNA synthetase fold. HisRS interacts in the same fashion with the histidine, adenosine and α-phosphate moieties of the substrates and intermediate, and GlyRS interacts in the same way with the adenosine and α-phosphate moieties in both states. In addition to the amino acid recognition, there is one key mechanistic difference between the two enzymes: HisRS uses an arginine whereas GlyRS employs a magnesium ion to catalyze the activation of the amino acid. ThrRS was complexed with its cognate tRNA and ATP, which represents the enzyme-substrate state of the second step of aminoacylation, i.e. the transfer of the amino acid to the 3′-terminal ribose of the tRNA. All three enzymes utilize class II conserved residues to interact with the adenosine-phosphate. ThrRS binds tRNA^Thr so that the acceptor stem enters the active site pocket above the adenylate, with the 3′-terminal OH positioned to pick up the amino acid, and the anticodon loop interacts with the C-terminal domain whose fold is shared by all three enzymes. We can thus extend the principles of tRNA binding to the other two enzymes

Achieving Error-Free Translation: The Mechanism of Proofreading of Threonyl-tRNA Synthetase at Atomic Resolution

The fidelity of aminoacylation of tRNAThr by the threonyl-tRNA synthetase (ThrRS) requires the discrimination of the cognate substrate threonine from the noncognate serine. Misacylation by serine is corrected in a proofreading or editing step. An editing site has been located 39 Å away from the aminoacylation site. We report the crystal structures of this editing domain in its apo form and in complex with the serine product, and with two nonhydrolyzable analogs of potential substrates: the terminal tRNA adenosine charged with serine, and seryl adenylate. The structures show how serine is recognized, and threonine rejected, and provide the structural basis for the editing mechanism, a water-mediated hydrolysis of the mischarged tRNA. When the adenylate analog binds in the editing site, a phosphate oxygen takes the place of one of the catalytic water molecules, thereby blocking the reaction. This rules out a correction mechanism that would occur before the binding of the amino acid on the tRNA

Modeling the Structure of RNA Molecules with Small-Angle X-Ray Scattering Data

<div><p>We propose a novel fragment assembly method for low-resolution modeling of RNA and show how it may be used along with small-angle X-ray solution scattering (SAXS) data to model low-resolution structures of particles having as many as 12 independent secondary structure elements. We assessed this model-building procedure by using both artificial data on a previously proposed benchmark and publicly available data. With the artificial data, SAXS-guided models show better similarity to native structures than ROSETTA decoys. The publicly available data showed that SAXS-guided models can be used to reinterpret RNA structures previously deposited in the Protein Data Bank. Our approach allows for fast and efficient building of <i>de novo</i> models of RNA using approximate secondary structures that can be readily obtained from existing bioinformatic approaches. We also offer a rigorous assessment of the resolving power of SAXS in the case of small RNA structures, along with a small multimetric benchmark of the proposed method.</p></div

Model of 1U8D against native structure and SAXS envelope.

<p>Comparison of the red model, and green native structure for the longest modeled RNA, 1U8D. Even though shape (grey) would seem a weak restraint, topology and contacts within the model structure correspond closely to the native (similarity between the model to either native, or reconstructed shape).</p