The rate at which many genes are expressed as proteins depends on a process called transcriptional elongation. This process takes place as the region of DNA that defines the gene is transcribed into an RNA molecule, and it is catalyzed by an enzyme called RNA polymerase II. However, this process often stalls shortly after it starts, and another enzyme called a positive transcription elongation factor is needed to restart it.

The human immunodeficiency virus (HIV) is a retrovirus that hijacks the gene expression machinery inside immune cells in order to replicate itself. To do this as efficiently as possible, the elongation factor needs to restart the transcription process as quickly as possible. To ensure that this happens the virus produces a protein called Tat that binds to the short region of RNA that has already been made. At the same time the Tat protein also combines with other proteins to form a multi-protein machine called the super elongation complex. Other proteins in the super elongation complex include a ‘scaffold’ protein called AFF4, a positive elongation factor called P-TEFb, and at least two additional transcription factors.

Until recently researchers did not know how the Tat protein was able to recruit super elongation complexes to the correct location without recruiting other complexes that contained similar protein subunits. Now Schulze-Gahmen et al. have shed new light on this mystery by working out the crystal structure of the complex formed by the elongation factor P-TEFb when it forms a complex with the Tat protein and a scaffold protein called AFF4.

The results show that direct interactions between the Tat and scaffold proteins help to recruit the super elongation complex to the correct location. The three-way interactions between Tat, AFF4, and P-TEFb form a binding surface that encourages the complex to bind to the RNA. Overall, Schulze-Gahmen et al. show that the super elongation complex is much more likely to be recognized by the Tat protein and then bind to RNA than just the elongation factor on its own.

Transcription of the HIV genome by RNA polymerase II (Pol II), like the expression of many cellular genes, is largely regulated at the step of transcript elongation. (Levine, 2011; Lin et al., 2011; Luo et al., 2012; Zhou et al., 2012). Pol II is recruited to the HIV promoter and initiates transcription, which stalls after a 30–50 nucleotide transcript containing the trans-activating response region (TAR) is formed. The HIV Tat protein bound to a host super elongation complex (SEC) recognizes TAR and releases the paused polymerase (He et al., 2010; Sobhian et al., 2010). It is yet unclear how TAR and Tat specifically recruit SECs in preference to other complexes in the cell that contain SEC subunits.

HIV Tat binds simultaneously to TAR and positive elongation factor b (P-TEFb), composed of CDK9 and Cyclin T1 (CycT1) subunits. In turn, P-TEFb and the transcriptional elongation factors ELL2 and ENL/AF9 associate with hydrophobic segments in the approximately 1200-amino-acid AFF1 or AFF4 scaffold, together forming the SEC (He et al., 2010; Lin et al., 2010; Sobhian et al., 2010). P-TEFb triggers promoter escape by phosphorylating two negative elongation factors (DSIF and NELF), as well as the C-terminal domain (CTD) of Pol II (Ott et al., 2011; Zhou et al., 2012; Lu et al., 2013). In contrast, ELL2 is thought to stimulate Pol II processivity (Shilatifard et al., 1997) and ENL/AF9 appears to bridge the SEC to RNA polymerase II-associated factor complexes (He et al., 2011). Overexpression of an AFF1 fragment that binds to P-TEFb, but not the other components of SECs, has a strong inhibitory effect on HIV transcription, indicating that productive HIV transcription requires a complete SEC (Lu et al., 2013a).

As a key regulator of transcription, P-TEFb itself is regulated through complex formation with other proteins and RNA. Recent studies have shown that the CycT1 subunit tightly associates with AFF1 or AFF4 and that the ternary complex of CDK9, CycT1, and AFF1 (He et al., 2010; Lin et al., 2010) moves between various active and inactive P-TEFb complexes (Lu et al., 2014). These assemblies include the SECs (He et al., 2010; Lin et al., 2010), a complex with Brd4 (Jang et al., 2005; Yang et al., 2005), and the inhibitory 7SK snRNP (Lu et al., 2013a; Yang et al., 2001; Yik et al., 2003). The existence of various P-TEFb complexes in the cell raises the question of how Tat and TAR discriminate among these functionally diverse assemblies.

An initial clue to the origin of the specificity of Tat and TAR for recognizing SECs was provided by the crystal structure of the AFF4-P-TEFb complex (Schulze-Gahmen et al., 2013). In this complex, the intrinsically disordered AFF4 fragment was folded on the surface of CycT1 in an orientation adjacent to the Tat binding site. A model of the quaternary Tat-AFF4-P-TEFb complex, based on the superposition of AFF4-P-TEFb and Tat-P-TEFb (Tahirov et al., 2010), predicted direct interactions between Tat and AFF4 (Schulze-Gahmen et al., 2013). These direct contacts were proposed to account for an 11-fold increase in the affinity of Tat for P-TEFb in the presence of AFF4.

To better understand the structural basis for the critical role of AFF1/4 in HIV transcription, we determined the crystal structure of P-TEFb in complex with Tat and AFF4. The Tat-AFF4-P-TEFb structure and in vivo reporter assays in HeLa cells confirm the direct Tat–AFF4 interactions. In addition, AFF4 contacts CycT1 residues that are part of the flexible Tat–TAR recognition motif (TRM) (Garber et al., 1998; Das et al., 2004), increasing the order of the TRM in the quaternary complex. The TRM wraps around Tat, exposing several basic residues, and contributing CycT1 C261 to coordinate a shared Zn²⁺ ion. Remarkably, AFF4 fragments containing the CycT1- and Tat-interacting segments increased the affinity of Tat-P-TEFb for TAR by 30-fold. These results support the idea that AFF4 contributes to TAR binding through two distinct and sequential mechanisms. AFF4 interactions with Tat directly favor SEC recruitment and AFF4 interactions that constrain the CycT1 TRM indirectly promote TAR binding.

The 2–73 fragment of the SEC scaffold protein, AFF4, binds with high affinity to P-TEFb and increases P-TEFb affinity for Tat 11-fold (Chou et al., 2013; Schulze-Gahmen et al., 2013). To define the structural basis for the increased affinity of the AFF4-P-TEFb complex for Tat, we determined the crystal structure of the quaternary complex of P-TEFb with AFF4_2–73 and Tat_1–57. The Tat 1–57 fragment is a minimal construct with high transcriptional activation (Garcia et al., 1988). The structure was determined using X-ray data to 3.0-Å resolution (R/R_free = 0.206/0.232; Figure 1A, Table 1) with three complexes in the asymmetric unit (a.u.).

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Two complexes related by a non-crystallographic two-fold rotation axis are nearly identical, while the third complex shows small differences due to different crystal contacts (Figure 1—figure supplement 1). For example, only the two dyad-related complexes show electron density for AFF4 helix 0 (residues 4–21, Figure 1A and Figure 1—figure supplement 3), which packs against αE and αI of the CDK9 subunit in the same complex and AFF4 35–39 from the two-fold-related complex in the a.u. Almost identical interactions between AFF4 helix 0 and CDK9 were observed in one out of three assemblies in the AFF4-P-TEFb structure (Schulze-Gahmen et al., 2013), in addition to crystal contacts between helix 0 and a crystallographically related CDK9 subunit. This recurrence of the same helical structure in different crystal environments suggests that AFF4 residues 4–21 prefer a helical conformation. The function of helix 0, however, remains in doubt because mutational effects on transcription do not match the AFF4 contacts in the interface, and stabilization of helix 0 depends on crystal packing (Schulze-Gahmen et al., 2013). We will focus on features shared among all complexes and point out differences when they are relevant for the discussion.

Tat binds in an extended conformation to AFF4-P-TEFb, with minor changes from the Tat-P-TEFb structure (Tahirov et al., 2010). The backbone of loop residues 27–30 shifts in response to contacts with CycT1 250–261 for two complexes in the a.u. Based on mass spectrometry and electron density, the baculovirus-expressed Tat, like Tat expressed in HEK293T cells (Jäger et al., 2012), is N-terminally acetylated (Figure 1—figure supplement 2). Acetylation of the Tat N-terminus removes a positive charge in the moderately hydrophobic Tat–CycT1 interface around Tat M1 and fills a cavity, probably leading to tighter anchoring of the Tat N-terminus to CycT1.

AFF4 residues 34- to 69-fold on the CycT1 surface, making multiple direct contacts with Tat K28, F32, and E2 (Figure 1) and burying Tat M1. The average size of the Tat–AFF4 interface is 305 Ų on Tat and 330 Ų on AFF4. The interactions between AFF4 and Tat are mostly hydrophobic and van der Waals contacts, but also include hydrogen bonds on each end of the interaction site (Figure 1A). The nexus of the Tat–AFF4 interface, Tat K28, is partially buried in a hydrophobic pocket formed by the side chains of AFF4 M62, F65, and Tat F32, and the main chain of AFF4 helix 2, residues 59–63. The Tat K28 side-chain amino group forms a hydrogen bond with the AFF4 E61 main-chain carbonyl at the pocket edge facing the solvent (Figure 1A). In addition, the Tat E2 side chain is positioned to form a hydrogen bond with the AFF4 D68 main-chain amide. The CycT1 TRM forms another side of the K28 pocket (Figure 1B).

Although AFF4 contains similar secondary structural elements observed in the absence of Tat (Schulze-Gahmen et al., 2013), coupled shifts occur to avoid collisions with Tat (Figure 2). Backbone RMS deviations in AFF4 residues 34–66 excluding the variable loop 43–45 range from 1.6 Å to 2.2 Å between AFF4-P-TEFb complexes with and without Tat. In contrast, RMS deviations for the same AFF4 residues of different complexes in the a. u. range from 0.32 Å to 0.58 Å. While AFF4 residues 34–40 coincide in the presence and absence of Tat, AFF4 helix 1 (residues 48–55) is shifted along the helix axis. This change positions AFF4 M55 to make contacts with L252 in the CycT1 TRM region (Figure 2A). AFF4 helix 2 (residues 58–66) is shifted away from the bound Tat and closer to the CycT1 surface formed by helices H2′, H3′ and the H3′–H4′ loop. This movement leads to the formation of additional hydrogen bonds between AFF4 and CycT1 in the Tat-AFF4-P-TEFb complex compared to the AFF4-P-TEFb complex. As a consequence of the shifts in the AFF4 backbone, unfavorable close contacts between AFF4 and Tat are avoided (Figure 2B).

Figure 2

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In contrast to the Tat-P-TEFb complex, in which the CycT1 TRM is disordered between residues 253 and 260, this functionally important segment is ordered in two conformations in the Tat-AFF4-P-TEFb complex (Figures 1, 3). In all three complexes in the a.u., P249 and N250 at the beginning of the TRM make multiple contacts with the main chain atoms at the C-terminal end of Tat helix 35–44. In addition, CycT1 L252 forms hydrophobic interactions with AFF4 M55 in all three complexes. The TRM structures start to diverge at this point. CycT1 residues 253–259 loop over Tat helix 28–33 in two conformations that converge at CycT1 C261 (Figure 3A, Figure 3—figure supplement 1). In the dyad-related complexes, the CycT1 TRM makes crystal contacts that include W258, R259, and A260, but in the third complex, the crystal contacts are restricted to the side chain of R251. In all complexes, W256 makes buried contacts with AFF4. Basic residues such as K253, R254, R259, and the polar N257, on the other hand, show continuous main-chain electron density but the exposed side chains are disordered. C261 binds the shared Tat Zn²⁺ ion in all three complexes. The presence of multiple conformations and relatively weak electron density for the TRM loop residues 253–260 indicates that this region is conformationally restrained but still quite flexible after Tat and AFF4 binding. These results suggest that in the presence of Tat, AFF4 partially orders the TRM (Figure 3B).

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The structure of the Tat-AFF4-P-TEFb complex points to AFF4 M62 and F65 as the major Tat-interacting residues. To test the contribution of the Tat–AFF4 interface to Tat-dependent transcription, we measured the effect of alanine substitutions on SEC recruitment and Tat-dependent HIV-1 transcription. These assays were performed with the AFF1 scaffold protein, because Tat has a stronger effect on HIV transcription with AFF1 than with AFF4 (He et al., 2010; Lu et al., 2014). AFF1 mutants V67A and F70A (corresponding to M62A and F65A in AFF4) were ectopically expressed in HeLa cells in the absence or presence of Tat(C22G), a mutation in the Zn²⁺ ion coordination site required for WT Tat activity (Garber et al., 1998). The Tat(C22G) mutation increases the dependence on AFF1 for efficient transactivation (Lu et al., 2014). Immunoprecipitation of tagged WT AFF1, as well as the V67A, F70A, and V67A/F70A variants, efficiently co-precipitated CDK9 and CycT1 (Figure 4A). In contrast, Tat(C22G) failed to co-precipitate P-TEFb (Figure 4B, lanes 1 & 2). This lack of binding was rescued by co-expressing WT AFF1 but not the three AFF1 alanine variants, V67A, F70A, and V67A/F70A (Figure 4B). These results suggest that AFF1 V67 and F70 are important for interactions between the scaffold and Tat. In turn, this interface stabilizes the AFF1-CycT1 association, as suggested by the Tat-AFF4-P-TEFb structure.

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The effects of changes in the Tat–AFF1 interface on Tat-dependent HIV transcription were measured using a HIV LTR-driven luciferase reporter system in HeLa-derived NH1 cells (He et al. 2010) that can express Tat(C22G). Although Tat(C22G) barely activated HIV transcription by itself (1.9-fold), this mutant strongly synergized with WT AFF1 to stimulate transcription to a much higher level (89-fold). Ectopic expression of WT AFF1 by itself only increased Tat-independent luciferase expression 13-fold (Figure 4C). In contrast, the AFF1 single (V67A and F70A) and double (V67A/F70A) alanine mutants showed significantly reduced cooperation with Tat(C22G) in activating HIV transcription compared to WT AFF1 (Figure 4C). Thus, the Tat–AFF4 interface is critical not only to enhance the binding of Tat to CycT1, but also to stimulate Tat-dependent HIV transcription.

The Tat–TAR recognition motif of CycT1 is essential for high affinity binding of P-TEFb-Tat to TAR (Garber et al., 1998). This critical segment (CycT1 250-264) at the C-terminal end of the cyclin domain interacts directly with TAR, as judged by RNA–protein cross-linking studies (Richter et al., 2002). Since AFF4 binds close to the TRM in the Tat-AFF4-P-TEFb structure, we investigated the effect of AFF4 on TAR binding. Electrophoretic mobility shift assays (EMSA) revealed unexpectedly that AFF4 fragments 32–67, 2–73, and 2–98 each increased the affinity of TAR for the Tat-P-TEFb complex by 30-fold (Figure 5A). It is unlikely that AFF4 directly contributes to TAR binding, since the AFF4-P-TEFb complex does not show any binding to TAR by itself. Instead, the Tat-AFF4-P-TEFb structure provides evidence that AFF4 binding in the presence of Tat restricts the conformational freedom of the CycT1 TRM region and positions this region for TAR interaction (Figure 5B).

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Efficient HIV transcription requires the Tat/TAR-mediated recruitment of SECs to the HIV promoter. Transcription remains stalled at the promoter in the absence of host elongation factors needed for Pol II to faithfully reach the distal end of the HIV genome. The X-ray structure of Tat-AFF4-P-TEFb reveals subunit interactions that mediate the preference of Tat for SECs over other P-TEFb complexes. As predicted (Schulze-Gahmen et al., 2013), Tat and AFF4 bind adjacent to each other on the CycT1 surface (Figure 1). The Tat–AFF4 interaction surface is centered around Tat K28, which is acetylated in vivo to regulate HIV transcription (Kiernan et al., 1999). Mutations in AFF1 corresponding to the Tat–AFF4 interface in the crystal structure reduce scaffold binding and transcription stimulation functions. Compared to the AFF4-P-TEFb complex, AFF4 structural segments in the Tat-AFF4-P-TEFb complex undergo unanticipated 1–2 Å backbone shifts to avoid unfavorable close contacts with Tat (Figure 2). These shifts in AFF4 alter the dimensions of the Tat-binding pocket in AFF4-P-TEFb that may serve as a site for targeting HIV transcription inhibitors (Schulze-Gahmen et al., 2013).

The Tat-AFF4-P-TEFb complex structure also reveals that the scaffold and Tat combine to partially fold the CycT1 TRM. This interaction is an example of multiple natively disordered protein segments coming together to form a structure that depends on the other protein subunits (Figure 3B). The conformations of the CycT1 TRM are strikingly different in the Tat-AFF4-P-TEFb complex compared to other structures containing P-TEFb. The TRM residues 253–260 are disordered in the Tat-P-TEFb crystal structure (Tahirov et al., 2010). In P-TEFb alone, crystal contacts stabilize the TRM in a distinct conformation that partially occludes the AFF4 binding site and precludes contacts between C261 and the Tat Zn²⁺ ion (Garber et al., 1998; Baumli et al., 2008).

The position of the TRM in the Tat-AFF4-P-TEFb structure, including exposed side chains for residues R251, R254, W258, and R259, is consistent with functional data (Garber et al., 1998). For example, alanine mutants of eight out of thirteen residues in the CycT1 segment 250–262 abolished or reduced in vitro binding of Tat-CycT1 to TAR. In addition, UV crosslinking of the CycT1 Tat–TAR complex (in the absence of AFF4) suggests that TAR makes direct contacts with the TRM (Richter et al., 2002).

The CycT1 TRM is not known to make RNA contacts during host transcription in uninfected cells, but the TRM contacts the loop in HIV TAR (Wei et al., 1998; Richter et al., 2002). To probe the complementarity of TAR with the Tat–CycT1 hybrid surface in the Tat-AFF4-P-TEFb complex structure, we manually docked the TAR-argininamide solution structure (PDB ID 1ARJ) (Puglisi et al., 1992; Aboul-ela et al., 1995) with Tat-AFF4-P-TEFb. Since, we could not model the side chains of CycT1 K253, R254, W258, and R259 in the electron density, we added these side chains in the most frequently observed conformation. The calculated electrostatic potential surface shows a large positively charged region covering the CycT1 TRM and part of Tat including R49 (Figure 5B). Positioning the TAR loop to contact the TRM enables the RNA bulge (U23–U25) to reach the Tat arginine rich motif (ARM) that makes essential contacts (Figure 5—figure supplement 1) (Weeks and Crothers, 1991). The dimensions of all components fit well and provide a plausible working model for the TAR interaction with Tat-AFF4-P-TEFb.

AFF4 not only promotes folding of the TRM, but the scaffold also increases TAR binding in vitro by 30-fold. This enhancement of RNA affinity is likely to be an indirect consequence of ordering the CycT1 TRM, because TAR is too small to make direct contacts with AFF4 residues in the complex. In Hela cells, alanine mutations in AFF1 (M60A/L61A) corresponding to the AFF4–TRM interaction site (M55/L56) abolish Tat(C22G)-SEC assembly and eliminate the inhibitory activity of AFF1 1-308 in Tat transactivation (Lu et al., 2014). Taken together, both the Tat-AFF4 interface and the TRM–AFF4 interaction are essential for WT Tat activity.

These results show that AFF4 contributes to the selective recruitment of SECs by Tat and TAR through a two-stage mechanism. First, direct interactions involving AFF4, Tat, and the TRM increase the binding affinity of Tat for AFF4-P-TEFb 11–fold (Schulze-Gahmen et al., 2013). Second, AFF4 binding to Tat-P-TEFb indirectly constrains the TRM conformation, increasing the binding affinity for TAR 30-fold. The enhancements of affinity in these successive steps together lead to a 330-fold increase in Tat/TAR binding by AFF4-P-TEFb over P-TEFb. This preference of Tat and TAR for the SECs, in concert with the Tat-stimulated release of scaffold-P-TEFb complexes from the 7SK snRNP (Lu et al., 2014), ensures preferential, simultaneous recruitment of the full complement of elongation factors required for efficient HIV transcription.

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Author details

1. Ursula Schulze-Gahmen

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   US-G, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   uschulze-gahmen@berkeley.edu

   Competing interests

   The authors declare that no competing interests exist.

2. Huasong Lu

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   HL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Qiang Zhou

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   QZ, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Tom Alber

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. California Institute for Quantitative Biosciences, QB3, University of California, Berkeley, Berkeley, United States

   Contribution

   TA, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

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