RESUMEN
Viruses manipulate multiple processes of the host cell machinery in order to replicate successfully in the infected cell. Among these, stimulation of transcription of the viral genes is crucial for lentiviruses such as HIV for increased protein expression levels and generation of escape mutants. The transactivation response (TAR) element at the 5'-end of HIV, SIV, BIV, EIAV or JDV retroviruses forms a unique RNA based promoter element that together with the transcription activator protein Tat stimulates viral gene expression at the level of transcription elongation. TAR is a double stranded non-coding RNA of typically 24-40 nucleotides length. Together with Tat it interacts with the Cyclin T subunit of the positive transcription elongation factor P-TEFb to recruit Cyclin T and its corresponding Cyclin-dependent kinase Cdk9 to the RNA polymerase II. In vitro formations of these Tat-TAR containing ribonucleoprotein complexes are a key requisite for biochemical characterizations and interaction studies that eventually will allow structural analyses. Here, we describe purification methods of the different factors employed and chromatography techniques that yield highly specific complex assemblies suitable for crystallization.
Asunto(s)
Duplicado del Terminal Largo de VIH , VIH-1/metabolismo , Ribonucleoproteínas/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cromatografía en Gel , Ciclina T/genética , Ciclina T/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , ARN no Traducido/química , ARN no Traducido/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/química , Alineación de Secuencia , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Positive transcription elongation factor b (P-TEFb) stimulates the transition from transcription initiation to productive elongation by phosphorylation of the C-terminal domain of RNA polymerase II. P-TEFb consists of the cyclin-dependent kinase Cdk9 and a T-type cyclin and is regulated by the small nuclear RNA 7SK and the coupling protein Hexim1 or Hexim2. In this study, we analyzed the tripartite protein-RNA complex formation between Hexim, Cyclin T and 7SK snRNA. Using isothermal titration calorimetry, we observed higher affinities for Cyclin T1-Hexim1 and Cyclin T2-Hexim2 complex formations compared with the interactions in reverse. Importin alpha, which is part of the Ran-mediated nuclear import pathway, bound Hexim1 and Hexim2 with dissociation constants of 2.0 and 0.5 muM, respectively. Furthermore, tripartite complex formations between Cyclin T, Hexim and Importin alpha showed the suitability of a collaborative nuclear import pathway for Cyclin T. Electrophoretic mobility shift assays using radioactively labelled full-length 7SK snRNA revealed a tight association of the RNA to Cyclin T1-Hexim1 with dissociation constants lower than 0.3 muM. Similar binding affinities were recorded for both Hexim orthologues to a 66-mer double-stranded 5' hairpin loop encompassing nucleotides 23-88 of 7SK, while a 39-mer fragment, resulting from different RNA folding predictions, did not bind as tightly. These results provide the molecular basis for the generation of a core complex for the inhibition of P-TEFb.
Asunto(s)
Ciclina T/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , alfa Carioferinas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Calorimetría , Cromatografía en Gel , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de Proteína , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Proteínas de Unión al ARN/química , Ribonucleoproteínas Nucleares Pequeñas/química , Termodinámica , Factores de TranscripciónRESUMEN
Brd4 is a member of the bromodomains and extra terminal domain (BET) family of proteins that recognize acetylated chromatin structures through their bromodomains and act as transcriptional activators. Brd4 functions as an associated factor and positive regulator of P-TEFb, a Cdk9-cyclin T heterodimer that stimulates transcriptional elongation by RNA polymerase II. Here, the crystal structures of the two bromodomains of Brd4 (BD1 and BD2) were determined at 1.5 and 1.2 A resolution, respectively. Complex formation of BD1 with a histone H3 tail polypeptide encompassing residues 12-19 showed binding of the Nzeta-acetylated lysine 14 to the conserved asparagine 140 of Brd4. In contrast, in BD2 the N-terminal linker sequence was found to interact with the binding site for acetylated lysines of the adjacent molecule to form continuous strings in the crystal lattice. This assembly shows for the first time a different binding ligand than acetylated lysine indicating that also other sequence compositions may be able to form similar interaction networks. Isothermal titration calorimetry revealed best binding of BD1 to H3 and of BD2 to H4 acetylated lysine sequences, suggesting alternating histone recognition specificities. Intriguingly, an acetylated lysine motif from cyclin T1 bound similarly well to BD2. Whereas the structure of Brd2 BD1 suggested its dimer formation, both Brd4 bromodomains appeared monomeric in solution as shown by size exclusion chromatography and mutational analyses.