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.
Highlights
At many genes in humans—including the integrated human immunodeficiency virus (HIV) genome as well as loci that regulate development and mediate responses to stress—RNA polymerase II initiates transcription but forms a stable paused complex after the synthesis of 30–50 nucleotides (Lin et al, 2011; Levine, 2012; Luo et al, 2012a; Zhou et al, 2012)
Tat increases the affinity of positive transcription elongation factor b (P-TEFb) for AFF4 by over an order of magnitude in vitro and rescues P-TEFb binding of AFF4 interface mutants in vivo. These results suggest that the super elongation complex (SEC) scaffold is an unanticipated direct partner of HIV-1 Tat, and an intersubunit Tat-binding pocket in the AFF4-P-TEFb complex may afford an unexpected site to target with selective inhibitors of HIV transcription
Consistent with the coupling of folding to binding, AFF4 lacks intramolecular tertiary contacts as it snakes across the cyclin T1 (CycT1) surface
Summary
At many genes in humans—including the integrated HIV genome as well as loci that regulate development and mediate responses to stress—RNA polymerase II initiates transcription but forms a stable paused complex after the synthesis of 30–50 nucleotides (Lin et al, 2011; Levine, 2012; Luo et al, 2012a; Zhou et al, 2012). These paused polymerases are poised for rapid, synchronous efficient transcription. Recruitment of active P-TEFb to the paused polymerase complex serves as an important checkpoint for gene expression (Levine, 2012; Luo et al, 2012b; Zhou et al, 2012)
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