Formation of Tat-TAR containing ribonucleoprotein complexes for biochemical and structural analyses.
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.
Bigalke
Janna M
JM
Max-Planck-Institut für molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Czudnochowski
Nadine
N
Vollmuth
Friederike
F
Vogel-Bachmayr
Karin
K
Anand
Kanchan
K
Geyer
Matthias
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D056906:Cyclin T / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D022661:RNA, Untranslated / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
D056906:Cyclin T; D022661:RNA, Untranslated; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D054322:tat Gene Products, Human Immunodeficiency Virus