title: A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat.
abstract: BACKGROUND
  The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding.
  
  
  RESULTS
  Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes.
  
  
  CONCLUSIONS
  Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription.
authors.forename: Nina
authors.initials: N
authors.lastname: Verstraete
authors.forename: Alona
authors.initials: A
authors.lastname: Kuzmina
authors.forename: Gaelle
authors.initials: G
authors.lastname: Diribarne
authors.forename: Van Trung
authors.initials: VT
authors.lastname: Nguyen
authors.forename: Lydia
authors.initials: L
authors.lastname: Kobbi
authors.forename: Monika
authors.initials: M
authors.lastname: Ludanyi
authors.forename: Ran
authors.initials: R
authors.lastname: Taube
authors.affiliation: Institut de Biologie de l'Ecole Normale Supérieure, Paris F-75005, France. bensaude@biologie.ens.fr.
authors.forename: Olivier
authors.initials: O
authors.lastname: Bensaude
mesh_terms: D056906:Cyclin T / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D017354:Point Mutation; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism*; D017510:Protein Folding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: C503684:CCNT1 protein, human; D056906:Cyclin T; C476123:HEXIM1 protein, human; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D042925:Positive Transcriptional Elongation Factor B