Formation of Tat–TAR containing ribonucleoprotein complexes for biochemical and structural analyses
| Autor: | Karin Vogel-Bachmayr, Friederike Vollmuth, Janna M. Bigalke, Nadine Czudnochowski, Kanchan Anand, Matthias Geyer |
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| Rok vydání: | 2011 |
| Předmět: |
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Molecular RNA Untranslated Cyclin T1 Recombinant Fusion Proteins viruses Molecular Sequence Data Electrophoretic Mobility Shift Assay RNA polymerase II Biology General Biochemistry Genetics and Molecular Biology 7SK RNA Amino Acid Sequence Protein Structure Quaternary P-TEFb Molecular Biology RNA polymerase II holoenzyme HIV Long Terminal Repeat Binding Sites Base Sequence General transcription factor Cyclin T Molecular biology Protein Structure Tertiary Cell biology Ribonucleoproteins Chromatography Gel HIV-1 biology.protein RNA Viral tat Gene Products Human Immunodeficiency Virus Transcription factor II D Sequence Alignment Transcription factor II B |
| Zdroj: | Methods. 53:78-84 |
| ISSN: | 1046-2023 |
| DOI: | 10.1016/j.ymeth.2010.04.001 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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