The AFF4 scaffold binds human P-TEFb adjacent to HIV Tat

Elife. 2013 Mar 5;2:e00327. doi: 10.7554/eLife.00327.

Abstract

Human positive transcription elongation factor b (P-TEFb) phosphorylates RNA polymerase II and regulatory proteins to trigger elongation of many gene transcripts. The HIV-1 Tat protein selectively recruits P-TEFb as part of a super elongation complex (SEC) organized on a flexible AFF1 or AFF4 scaffold. To understand this specificity and determine if scaffold binding alters P-TEFb conformation, we determined the structure of a tripartite complex containing the recognition regions of P-TEFb and AFF4. AFF4 meanders over the surface of the P-TEFb cyclin T1 (CycT1) subunit but makes no stable contacts with the CDK9 kinase subunit. Interface mutations reduced CycT1 binding and AFF4-dependent transcription. AFF4 is positioned to make unexpected direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. These studies define the mechanism of scaffold recognition by P-TEFb and reveal an unanticipated intersubunit pocket on the AFF4 SEC that potentially represents a target for therapeutic intervention against HIV/AIDS. DOI:http://dx.doi.org/10.7554/eLife.00327.001.

Keywords: Human; SEC; intrinsically disordered proteins; super elongation complex; transcription elongation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Crystallography, X-Ray
  • Cyclin T / metabolism
  • Cyclin-Dependent Kinase 9 / metabolism
  • Gene Expression Regulation, Viral
  • HIV-1 / genetics
  • HIV-1 / growth & development
  • HIV-1 / metabolism*
  • Humans
  • Models, Molecular
  • Positive Transcriptional Elongation Factor B / chemistry
  • Positive Transcriptional Elongation Factor B / metabolism*
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Recombinant Proteins / metabolism
  • Repressor Proteins / chemistry
  • Repressor Proteins / metabolism*
  • Transcription Elongation, Genetic
  • Transcriptional Elongation Factors
  • Virus Replication
  • tat Gene Products, Human Immunodeficiency Virus / chemistry
  • tat Gene Products, Human Immunodeficiency Virus / metabolism*

Substances

  • AFF4 protein, human
  • CCNT1 protein, human
  • Cyclin T
  • Recombinant Proteins
  • Repressor Proteins
  • Transcriptional Elongation Factors
  • tat Gene Products, Human Immunodeficiency Virus
  • Positive Transcriptional Elongation Factor B
  • CDK9 protein, human
  • Cyclin-Dependent Kinase 9