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

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

AFF1 is a ubiquitous P-TEFb partner to enable Tat extraction of P-TEFb from 7SK snRNP and formation of SECs for HIV transactivation

“…Meanwhile, more AFF1 became associated with P-TEFb in the presence of Tat than in its absence (Fig. 3A, compare lanes 3 and 5), as is consistent with the previous demonstration that Tat enhances the affinity of the AFF1 homolog AFF4 for P-TEFb (12).…”

“…With AFF1 shown to be a bona fide subunit of 7SK snRNP, it is important to determine the functional significance of this phenomenon. The recently solved AFF4-P-TEFb crystal structure reveals that the AFF1 homolog AFF4 is positioned to make direct contacts with HIV Tat on the surface of CycT1 (12). In light of this information and the previous demonstrations that Tat is able to extract P-TEFb directly from 7SK snRNP both in vitro and in HIV-infected cells (7), we examined the effect of AFF1 on Tat-induced disruption of 7SK snRNP.…”

“…2B). Moreover, upon fusion to GFP and the nuclear localization sequence, two shorter AFF1 fragments, amino acids 1-77 and 38-72, which correspond to the biochemically identified minimal CBS of AFF4 (amino acids 2-73) (11) and the ordered AFF4 segment resolved in the AFF4-P-TEFb crystal structure (12), respectively, were tested also (Fig. 2C).…”

“…Several di-alanine substitutions introduced into the AFF4 CBS have been shown to decrease AFF4's binding to P-TEFb and to inhibit basal HIV transcription (11,12). Because the CBS also is important for AFF1's interaction with 7SK snRNP, we generated and tested three AFF1 di-alanine mutations based on the effects of their corresponding mutations in AFF4.…”

“…Within the SEC, the AF4/FMR2 family proteins AFF1 and AFF4 are characterized as highly flexible scaffolding subunits that use short hydrophobic regions along their structurally disordered axis to bring two different transcription elongation factors, P-TEFb and ELL1 or 2, and members of the elevennineteen-leukemia/ALL1-fused gene from chromosome 9 protein (ENL/AF9) family into a single complex (10,11). The recently solved AFF4-P-TEFb crystal structure further illustrates that a short sequence (amino acids 34-67) located near the AFF4 N terminus snakes across the surface of CycT1 and also may make direct contacts with Tat (12). When an SEC is recruited to the HIV promoter by Tat and TAR, the contained P-TEFb and ELL1/2 components potentially can act on the same polymerase enzyme to activate transcription synergistically (10).…”