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The 7SK small nuclear RNA (7SKsnRNA) plays a key role in the regulation of RNA polymerase II by sequestrating and inhibiting the positive transcription elongation factor b (P-TEFb) in the 7SK ribonucleoprotein complex (7SKsnRNP), a process mediated by interaction with the protein HEXIM. P-TEFb is also an essential cellular factor recruited by the viral protein Tat to ensure the replication of the viral RNA in the infection cycle of the human immunodeficiency virus (HIV-1). Tat promotes the release of P-TEFb from the 7SKsnRNP and subsequent activation of transcription, by displacing HEXIM from the 5'-hairpin of the 7SKsnRNA. This hairpin (HP1), comprising the signature sequence of the 7SKsnRNA, has been the subject of three independent structural studies aimed at identifying the structural features that could drive the recognition by the two proteins, both depending on arginine-rich motifs (ARM). Interestingly, four distinct structures were determined. In an attempt to provide a comprehensive view of the structure-function relationship of this versatile RNA, we present here a structural analysis of the models, highlighting how HP1 is able to adopt distinct conformations with significant impact on the compactness of the molecule. Since these models are solved under different conditions by nuclear magnetic resonance (NMR) and crystallography, the impact of the buffer composition on the conformational variation was investigated by complementary biophysical approaches. Finally, using isothermal titration calorimetry, we determined the thermodynamic signatures of the Tat-ARM and HEXIM-ARM peptide interactions with the RNA, showing that they are associated with distinct binding mechanisms.

The small nuclear 7SK RNA regulates RNA polymerase II (RNA Pol II) transcription, by sequestering and inhibiting the positive transcription elongation factor b (P-TEFb). P-TEFb is stored in the 7SK ribonucleoprotein (RNP) that contains the three nuclear proteins Hexim1, LaRP7, and MePCE. P-TEFb interacts with the protein Hexim1 and the 7SK RNA. Once P-TEFb is released from the 7SK RNP, it activates transcription by phosphorylating the C-terminal domain of RNA Pol II. P-TEFb also plays a crucial role in the replication of the human immunodeficiency virus HIV-1, through its recruitment by the viral transactivator Tat. Previous work demonstrated that the protein Tat promotes the release of P-TEFb from the 7SK RNP through direct binding to the 7SK RNA. Hexim1 and Tat proteins both comprise conserved and similar arginine-rich motifs that were identified to bind the 7SK RNA at a repeated GAUC site located at the top of the 5′-terminal hairpin (HPI). Here, we report the solution structure of this region as determined by nuclear magnetic resonance, to identify HPI structural features recognized by Hexim1 and Tat. The HPI solution structure displays an elongated shape featuring four helical segments interrupted by one internal loop and three bulges with distinct folds. In particular, the repeated GAUC motif adopts a pre-organized geometry. Our results suggest that the binding of Hexim1 and Tat to the 7SK RNA could originate from a conformational selection of this motif, highlighting how RNA local structure could lead to an adaptive recognition of their partners.

Aptamers are oligonucleotides, generally DNA or RNA but occasionnally chemically modified nucleic acids, which are identified within randomly synthesized libraries containing up to 1015 different candidates. They are obtained following a process of in vitro selection termed systematic evolution of ligands by exponantial enrichment (SELEX) that makes use of iterative steps of selection and amplification. Aptamers were successfully raised against a wide range of targets: amino acids, antibiotics, dyes, peptides, proteins, nucleic acids, intact viruses or live cells. They generally display high affinity (Kd in the nanomolar range or lower are frequent for proteins) and high specificity. They are easily obtained by chemical synthesis and can be converted in tools of interest for diagnostic purposes, by conjugation to various pendant groups. They rival antibodies and can be used in vivo in human beings.

AbstractMichael addition of alkoxides or diethyl methylmalonate to 2-chloroacrylonitrile provides functional carbanions which are able to react with 1,3-dinitrobenzene following a vicarious nucleophilic substitution to give new substituted nitroarenes.

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