HEXIM1 inter-monomer autoinhibition governs 7SK RNA binding specificity and P-TEFb inactivation

Domain: chemistry
Published: 2026-01-15
Editorial outcome: minor_revision

Abstract
Hexim proteins are key RNA-dependent regulators of eukaryotic transcription through 7SK-dependent sequestration and inactivation of the kinase P-TEFb (Cdk9–Cyclin T1/2) in the 7SK RNP. P-TEFb activity drives release of RNA polymerase II from promoter-proximal pausing for eukaryotic and HIV-1 transcription. The molecular mechanism by which 7SK binding overcomes an intrinsic Hexim autoinhibition for subsequent P-TEFb inactivation has remained unresolved. Here, using NMR and biophysical methods we demonstrate that Hexim1 homodimer engages two high-affinity sites on 7SK RNA. This dual-site binding triggers a conformational rearrangement in Hexim1’s disordered central region that unmasks the Cdk9-binding site, which is otherwise sequestered within an inter-monomer dimer interface. These findings reveal how Hexim autoinhibition dictates its specificity for 7SK RNA and prevents premature P-TEFb inhibition in the absence of 7SK, thereby providing a mechanistic understanding of Hexim/P-TEFb assembly into the 7SK RNP and further considerations for understanding Hexim–Tat competition during viral transcription.

Introduction
HEXIM (HEXamethylene-bis-acetamide-Inducible protein in vascular smooth Muscle cells)1 is a unique family of RNA-dependent regulatory proteins2 whose principal target is 7SK RNA, an abundant ~332-nucleotide non-coding RNA that plays a central role in regulating RNA polymerase II (Pol II) transcription. In addition to 7SK, Hexim has been shown to interact with other RNAs, including NEAT13 and several mRNAs4, hinting at broader cellular functions. Mammals express two paralogs of Hexim (Hexim1 and Hexim2) which differ in tissue distribution and can functionally compensate for one another within the 7SK ribonucleoprotein complex (7SK RNP)5,6. Hexim suppresses the kinase activity of positive transcription elongation factor b (P-TEFb) by sequestering it within the 7SK RNP. Crucially, this sequestration depends on 7SK RNA binding to Hexim7–9. P-TEFb is a heterodimer comprising the catalytic subunit Cdk9 and a regulatory subunit, Cyclin T1 or T2 (CycT1/CycT2), which exhibits specificity for Hexim1 and Hexim2, respectively10. Treatment of live cells with a Cdk9 inhibitor mobilizes hnRNPs to bind to 7SK and consequently evict both Hexim and P-TEFb, not associated with each other, from 7SK within minutes11. Upon release from 7SK RNP, P-TEFb phosphorylates the C-terminal domain (CTD) of Pol II along with several other transcription factors (NELF, DSIF, PAF, SPT6), a process essential for Pol II to transition from promoter-proximal paused to productive elongation12,13. Beyond its host regulatory roles, 7SK RNP is also a critical target for HIV-1 replication, where Tat (Trans-activator of transcription) protein hijacks the 7SK RNP to the HIV-1 promoter and extracts P-TEFb into a viral super elongation complex via the nascent TAR (trans-activation response) RNA element14–17.

Hexim1 contains three main regions: (i) the N-terminal region (1-149), which is poorly conserved across vertebrates (Supplementary Figs. 1 and 2) and predicted to be intrinsically disordered (Supplementary Fig. 3), (ii) the central region (150-253), discussed below, and (iii) a C-terminal coiled-coil domain (CC hereafter, 254-359) that has been shown by NMR to form a stable homodimer18 and mediates Hexim1 binding to CycT119 (Fig. 1A). Within the central region only the Basic Region (BR) has been structurally characterized7,8. BR directly interacts with RNA and comprises two Arg/Lys-rich stretches separated by a conserved proline-serine and is often referred to as ARM (arginine-rich motif)7,20 due to its sequence similarity to the single ARM of HIV-1 Tat (Fig. 1B). Following the BR is a linker containing a conserved PYNT motif, known to bind Cdk97,21 and act in concert with the CC to inactivate P-TEFb9 (Fig. 1A). The linker is followed by an Acidic Region (AR). A prevailing model proposes that an internal electrostatic interaction between the BR and AR maintains Hexim in an autoinhibited state that cannot bind P-TEFb, rendering P-TEFb inhibition RNA-dependent9,22. However, the molecular mechanism by which this autoinhibition prevents PYNT–Cdk9 engagement in the absence of RNA remains unknown.

Hexim–RNA binding specificity and stoichiometry have long been subjects of debate. One study suggested Hexim1 acts as a general binder of double-stranded RNA23, whereas structural studies24–27 have focused on a specific motif—a GAUC palindrome—within stem-loop 1 (SL1) of 7SK RNA. This site (Site 1 in Fig. 1C), encompassing GAUC2 and GAUC3 motifs (nts 42-45 and 64-67), is a conserved marker for 7SK genes28,29 and also serves as the RNA-binding site for HIV-1 Tat26,30. However, a biochemical study30 identified a second Hexim1 binding site in SL1 lacking a GAUC motif (Site 2 in Fig. 1C). Lastly, another GAUC motif (GAUC1, nts 13-16) is present in proximal SL1 within one RNA helical turn of the binding/capping site for methyl phosphate capping enzyme (MePCE)31.

The issues of Hexim binding specificity and stoichiometry are further complicated by evidence32,33, most recently from structural studies34 and RNA chemical probing by DANCE-MaP35, of at least two distinct 7SK RNA conformations in vitro and in cells: a linear form, where a long SL1 presents the Hexim binding sites discussed above, and a circular form, in which a shorter SL1alt forms a rearranged GAUC palindrome (GAUC1 and GAUC2; Site 3 in Fig. 1C). Two core components, MePCE and Larp7, constitutively assemble with 7SK RNA36 for both linear and circular conformations, as shown by cryo-EM structures34 (Fig. 1C). Notably, switching between P-TEFb-bound and -free 7SK RNP pools, likely corresponding to the two RNA conformations, is required for stress-induced regulation of P-TEFb release34,35,37.

Results
Intrinsically disordered Hexim1 BR-L-AR is prone to intermolecular interaction
To characterize the Hexim1 central region, we expressed and purified a monomeric construct (residues 142–253) encompassing BR, linker (L) containing PYNT motif, and AR (hereafter referred to as BR-L-AR). Several additionally truncated Hexim1 monomeric constructs, BRARM1, BR, and BR-L (Fig. 1D), were used to compare with BR-L-AR. We observed a solubility limit of ~70 μM for BR-L-AR despite testing many conditions. In contrast, BRARM1, BR, and BR-L are easily soluble at ~0.5–0.8 mM under similar conditions. This suggests that BR-L-AR is prone to aggregation, mediated by intermolecular interactions that require both BR and AR. To detect the species formed via intermolecular interactions, we used native polyacrylamide gel electrophoresis (Native PAGE38) and mass photometry39,40 at ~4–25 μM and 40 nM, respectively. A clear ladder pattern of monomer and various oligomers was observed in Native PAGE for BR-L-AR, and mass photometry data captured trimer and tetramer species (Supplementary Fig. 4A–C and Fig. 1E). This indicates that the intermolecular interaction of BR-L-AR is stable enough to be observed even at 40 nM.

In contrast, freshly purified Hexim1 and L-AR-CC dimeric proteins both migrated as a single major band in Native PAGE, and mass photometry showed a homogeneous particle distribution corresponding to dimers (Fig. 1A, D and Supplementary Fig. 4A, D, E). However, when we combined BR-L-AR with either dimeric protein, we observed a stable intermolecular complex by mass photometry (Fig. 1E and Supplementary Fig. 4D, E). Together, these data suggest that in full-length Hexim1, the BR-L-AR from each monomer interact with each other, consistent with the previous model that interaction between BR and AR maintains Hexim in an autoinhibited state9,22. These interactions are robust enough to prevent oligomerization of the dimeric Hexim1, but are readily competed by adding BR-L-AR monomeric protein in trans, suggesting relatively fast exchange dynamics, consistent with the nature of interactions formed by charged disordered protein regions (IDR)41.

To determine the extent of disorder and secondary structure in BR-L-AR, we used multi-dimensional heteronuclear NMR experiments on uniformly 15N, 13C-labeled protein (see “Methods”). Despite the low protein BR-L-AR concentration (<70 μM) we could achieve due to intermolecular interactions, we were able to assign 1H, 15N, 1Hα, 13Cα, and 13Cβ chemical shifts and calculate secondary structure propensity (SSP) scores42 using Cα and Cβ chemical shifts (Fig. 1F and Supplementary Fig. 5A). The SSP scores, ranging from +1 to −1, reflect the fraction of α- or β-structure formed by each residue. From the SSP scores, we identified an α-helix (aa 171–194) in the linker region, between BR and PYNT motif (Fig. 1F). We also defined the two Arg/Lys-rich regions in BR as ARM1 and ARM2 (Fig. 1C): of these, ARM1 (residues 151–154) shows a moderate propensity to form β-structure, with a minimum SSP reaching −0.45. The rest of BR-L-AR is largely disordered, with SSP scores within +0.3 to −0.3 (Fig. 1F).

In summary, BR-L-AR is largely intrinsically disordered, contains a short α-helix in the linker following the BR, and has a strong tendency to form intermolecular interactions with other monomers and with Hexim1 dimer.

Characterization of Hexim monomer–7SK RNP Site 1 interaction
To look at the interaction of BR-L-AR with the previously characterized single 7SK RNA binding site (SL1 Site 1 in Fig. 1C)24–27,43,44, we first investigated the structure of two designed RNA constructs from the distal end of linear 7SK SL1 (SL1-dI and SL1-dIΔU63) using NMR (Fig. 2A, C). We assigned N-H…N imino (base-paired U/G) and non-exchangeable base and H1′ proton (all nucleotides) resonances for SL1-dI (Supplementary Figs. 6–8). Our analysis confirmed its complex architecture, which consists of a base-paired stem flanked by two dynamic U-rich bulges, consistent with previous structural studies24–27,43,44 (Fig. 2A). Those studies had variously reported that the two flanking U-rich bulges (U40U41 and U63) can form base triples with the A43-U66 and U65-A44 or G42-C67 base pairs, respectively; however, which of these base triples were observed varied with sample conditions including pH25,26,43,44. For SL1-dI, at 150 mM KCl and pH 6.2, we observed a stable U40…A43-U66 base-triple and a dynamic U63…A65-U44, where the U63 bulge samples at least two states, likely between stacking on G64 and Hoogsteen pairing with A65 (Fig. 2A and Supplementary Fig. 6A, C). To investigate the relative dynamics of these nucleotides, the non-exchangeable resonance intensities from non-constant time 13C-1H HQSC spectra were acquired, and normalized 13C intensities were plotted, where higher normalized intensity generally reflects faster dynamics on the pico-to-nanosecond timescale45 (Fig. 2A). These measurements confirmed that U40 is stable (low intensities of 0.1 as helical stem) while U41, U63, and A65 are much more flexible (elevated intensities) consistent with our observation of one stable and one dynamic base triple (Fig. 2A). At pH 5.2, both these base triples were stably observed26.

Next, we titrated BR-L-AR into the RNAs and monitored chemical shift perturbations (CSP). On the RNA side, for SL1-dI, the entire GAUC2-GAUC3 stem, U40U41 and U63 bulges are significantly shifted, with G64 affected the most (Fig. 2B, C and Supplementary Fig. 6). For SL1-dIΔU63, although the same overall Site 1 was used for binding, the distal half of GAUC2-GAUC3 stem exhibits smaller CSP (Fig. 2C and Supplementary Fig. 8), indicating a moderate contribution of U63 bulge (deleted in SL1-dIΔU63) to Hexim1 binding, consistent with an EMSA binding assay44. On the protein side, 15N-1H HSQC spectra of BR-L-AR titrated with either SL1-dIΔU63 or SL1-dI showed significant chemical shift changes and severe peak broadening for protein residues 151-170 spanning the entire BR (including ARM1, ARM2, and surrounding sequence) (Fig. 2D, E and Supplementary Fig. 9). Less line broadening is observed for BR-L-AR when SL1-dIΔU63 is added than for SL1-dI, indicative of a shift towards fast exchange on the NMR timescale likely due to a faster off-rate46. This allowed better tracing of the peak migration trajectories and observation of the BR residue S158 in the RNA-bound spectrum to confirm the binding site (Fig. 2D, E). Interestingly, in both cases, the C-terminal region of AR (c-AR) exhibited chemical shift changes at residues Phe241-Met242. To verify that the c-AR shift is indirect, we monitored NMR titration of SL1-dIΔU63 into the shorter BR-L or BR and found that it bound the same way as BR-L-AR (Supplementary Fig. 10A–E). These results indicate 7SK RNA binding to BR induces an allosteric change in c-AR.

In summary, 7SK SL1 Site 1 features a stem with two flanking bulges that can form one stable and one dynamic U…A-U base-triple, and both base-triples are required for specific BR binding that engages the entire GAUC-palindrome. Hexim1 uses both ARM1 and ARM2 of BR for RNA binding, and this binding induces a conformational change in BR-L-AR. In contrast to Hexim, HIV-1 Tat protein has only ARM1 that is necessary and sufficient for binding Site 1 (Fig. 1C)25,26,30, indicating a significant difference in Hexim and Tat recognition of 7SK RNA.

Characterization of Hexim1 autoinhibited conformation by PRE
To characterize the autoinhibitory interactions within the BR-L-AR region, we used Paramagnetic Relaxation Enhancement (PRE)47 NMR. This method can map weak and transient interactions48,49 common in IDRs50 by measuring distance-dependent line broadening from a covalently attached MTSL nitroxide spin label up to 25 Å away. A key challenge is distinguishing intra-monomer interactions from inter-monomer interactions. To dissect these, we established two distinct experimental setups using three individual cysteine substitution sites (S226C, S233C, S237C): (i) “total interaction” experiment: 15N-labeled and MTSL-tagged BR-L-AR, capturing both intra- and inter-monomer contacts (Fig. 3A, B and Supplementary Fig. 11); and (ii) “inter-only” experiment: 1:1 mixture of the reporter 15N-labeled, untagged BR-L-AR with the broadener 14N-labeled and MTSL-tagged BR-L-AR (Fig. 3C–E). In the latter, only inter-monomer interactions will result in line-broadening, however to a lesser extent than the “total” experiments, since only 50% of the “dimer” population is the combination of 15N and 14N-MTSL labeled proteins. To visualize and identify interactions between structural segments, we defined regions of primary broadening (red) and secondary broadening (orange) as stretches harboring 3 or more residues (either contiguous or non-contiguous) with intensity ratios lower than [mean - σ] and lower than [mean - 0.5xσ], respectively (Fig. 3B–E).

In the inter-only experiments, we observed a distinct pattern: S237CMTSL primarily broadens the entire BR (154-168), with ARM2 broadened more severely than ARM1 (Fig. 3C); S233CMTSL broadens ARM2-PYYK (Fig. 3D); and S226CMTSL broadens ARM2-PYYK, beginning of αL, and PYNT motif (Fig. 3E). This pattern indicates an overall head-to-tail orientation for BR-L-AR inter-monomer interaction (Fig. 3F), where the primary broadened region (red) shifts in the N- to C-terminal direction when we move the MTSL-tag from the C- to N-terminal direction (dashed lines in Fig. 3C–E).

We then analyzed the “total interaction” experiment by comparing this “total” profile to the “inter-only” S226C profile (compare Figs. 3B–E) to deconvolve the two interaction types. The secondary broadening (orange) observed in the “total” experiment at ARM2-PYYK and PYNT motif matched the “inter-only” experiment, confirming this as the inter-monomer component. Therefore, the remaining primary broadening, which was observed at both the beginning and the end of the αL, represents the intra-monomer component (Fig. 3B, F). This indicates that helix αL is bent or kinked, allowing its two ends to approach S226C. In the S237CMTSL “total” experiment, almost all the resonances are broadened more severely than S226CMTSL (Supplementary Fig. 11), indicating a significant role of c-AR in the autoinhibition, consistent with Phe241-Met242 exhibiting allosteric CSP upon RNA binding (Fig. 2E).

In summary, PRE NMR experiments distinguished and mapped intra- and inter-monomer interactions in Hexim1 BR-L-AR. Based on these results, the inter-monomer interaction adopts a head-to-tail orientation (N→C to C→N), which in full-length Hexim1 would correspond to a self-hugging topology between the two monomers (Fig. 3F).

PYNT motif is sequestered by autoinhibition and is unmasked upon RNA binding
The PYNT motif (conserved residues 202PYNTTQFLM210) plays a critical role in directly binding and inactivating the Cdk9 kinase subunit of P-TEFb7,21. We further investigated the PYNT interactions in BR-L-AR using an A201CMTSL substitution which positions the MTSL-tag immediately N-terminal to the PYNT motif (Fig. 4A). The PRE profile of 15N BR-L-AR(A201CMTSL) showed significant broadening of the PYNT motif itself, BR and c-AR (Fig. 4A and Supplementary Fig. 12). This result recapitulated the findings from BR-L-AR(S226CMTSL) (Fig. 3B orange) and confirms that the PYNT motif is a central node in the BR–PYNT–AR inter-monomer network. Based on data suggesting that αL is involved (Fig. 3C), we next probed from its central region using a S183CMTSL tag (Supplementary Fig. 13). The PRE profile of 15N BR-L-AR(S183CMTSL) showed significant broadening extending from the αL through the PYNT motif excluding the C-terminal end of αL, which along with BR and the two ends of AR shows secondary broadening (Fig. 4B). This indicates that the inter-monomer BR–PYNT–AR interactions partially involve the central region of αL.

Lastly, we investigated the effect of 7SK RNA binding on the inter-monomer interactions. Addition of SL1-dI to 15N BR-L-AR(S183CMTSL) resulted in significant peak intensity recovery across the entire protein, except for BR (Fig. 4B, cyan bars). This recovery signifies that the PRE-induced broadening has disappeared, meaning the inter-monomer network has been disrupted and the S183CMTSL tag is no longer in proximity to the rest of the protein. Note that the lack of intensity recovery for the BR is expected. As shown previously (Supplementary Fig. 9), BR residues are independently and severely broadened by the direct binding of SL1-dI RNA, masking any PRE-related effects. Taken together, the PRE data show that the PYNT motif directly participates in inter-monomer interactions, where it simultaneously contacts both BR and AR. This autoinhibitory sequestration is released upon BR binding to RNA, resulting in the unmasking of PYNT, making it available to bind and inactivate Cdk9 in the 7SK RNP.

Linear 7SK SL1 contains two high-affinity BR binding sites
While binding of Hexim1 BR to SL1 Site 1 has been well documented, other potential binding sites have been proposed but not characterized24–26,30,43,44. To resolve the outstanding questions of Hexim1–7SK RNA binding site location(s), stoichiometry, and specificity, we systematically investigated the full 7SK SL1 domain in linear versus circular conformations. Linear 7SK SL1 folds into a minimum 36-bp long stem containing eight internal bulges or loops (Fig. 5A). We employed a “divide-and-conquer” strategy, designing a series of RNA stem-loop segments from the distal (d), middle (m) and proximal (p) regions of linear SL1, as well as circular SL1alt. All constructs include a minimum 4-bp stem with one flanking bulge, based on the Hexim1 binding requirements for Site 1 described above. We then used NMR chemical shift perturbations (CSPs) to map binding sites (Fig. 5) and EMSA to determine stoichiometry (Supplementary Figs. 14, 15). For NMR mapping, BRARM1 was used in order to reduce the line broadening in the longer RNA constructs (SL1-d, dIIm, p and mp) when bound to Hexim1 protein. We verified that the same nucleotides in Site 1 were perturbed for BRARM1 as for BR-L-AR (Fig. 5 and Supplementary Fig. 17D). NMR assignments, CSP plots and 1D spectra of titration series for the additional RNA constructs studied (Supplementary Figs. 16–22) were acquired as for Site 1. Using the RNA CSPs (Fig. 5A–D), we mapped binding Sites 1, 2, 4 and 5 in linear SL1 and Site 3 in circular SL1alt. The non-specific site at SL1-pII exhibits minimum CSP upon binding BRARM1, providing a negative control. The binding sites are numbered based on the relative order of binding affinities observed by NMR, where Site 1 (not Site 4) shifted by adding 1:1 BRARM1 to SL1-d, Site 2 (not Site 4) shifted in SL1-dIIm, and Site 2 (not Site 5) shifted in SL1-mp (Fig. 5A); and by EMSA experiments, where two sequential shifts were observed for SL1-dΔU63 and SL1-d, corresponding to the high-affinity Site 1 followed by low-affinity Site 4 (Supplementary Fig. 14D, E and Notes). The largest CSP values and the exchange behaviors on the NMR timescale also agreed well with the order of binding affinities determined from combined NMR and EMSA analysis (Fig. 5A stars and Supplementary Fig. 17C).

To quantitatively compare the binding affinities of these RNAs, we acquired thermodynamic binding parameters by ITC (Figs. 5E, 6). Due to sample precipitation that occurs during ITC titration of RNA into BR-L-AR, potentially due to stirring, we instead used N-BR-L-AR (NBLA hereafter) that includes the N-terminal region. ITC measurements yielded KD values of 83, 189, and 261 nM for Sites 1, 2 and 3, respectively. Together, these findings indicate that linear 7SK SL1 contains two high-affinity sites and two low-affinity sites for BR. In contrast, circular 7SK SL1alt contains only one binding site for one BR, at the alternative GAUC1-GAUC2 palindrome (Fig. 5D). Comparison of these 5 binding sites indicates that the determining factor for high-affinity Hexim1 binding appears to be an A-U rich stem with at least one flanking 5′ U-containing bulge that participates in a U…A-U base triple, a common feature for Sites 1 and 2, but not for Sites 4 and 5, or the non-specific site SL1-pII. Site 3 can form a U…A-U base triple, but the U bulge is now on the 3′ side of the GAUC1-GAUC2 stem instead of on the 5′ side in Site 1, geometrically analogous to the U bulge of Site 1, consistent with a lower affinity than Sites 1 and 2.

S158 phosphorylation minimally affects autoinhibition and S158EE impairs RNA binding
Multiple phosphorylation sites have been identified in Hexim1 unstructured regions and suggested to play roles in releasing P-TEFb from the 7SK RNP complex51–54, including phosphorylation of BR residue Ser158 (pSer158) by protein kinase C (PKC)55 or protein kinase A (PKA)56. To investigate the effect of pSer158 on Hexim1 conformation, we phosphorylated BR-L-AR in vitro using PKA catalytic subunit (PKAcat) and monitored phosphorylation by NMR 1H-15N HSQC (Fig. 6A, B). A large shift of S158 amide resonance was observed due to pS158, with induced shifts at adjacent residues 159–161 and R156 (P157 lacks an amide) (Fig. 6A, C). Of note, c-AR residues F241 and M242 also show small chemical shift changes, although to a much smaller extent (CSP ~10-fold smaller than in Fig. 2C). This suggests that pS158 only minimally induces the same allosteric change in c-AR as RNA binding does, largely maintaining autoinhibition.

Longer incubation with PKA resulted in phosphorylation of additional sites (T171 and S183) and destabilization of the entire αL (Supplementary Fig. 23A). We note that αL does contribute to RNA binding affinity, as shown by our EMSA (compare Supplementary Fig. 14C to 14A). To avoid potential destabilization of αL and assure a homogenous sample, as well as to avoid a predicted pS49 by PKA (Supplementary Fig. 23B), we made a phosphomimetic variant NBLA-S158EE to look at the effect of two additional negative charges in BR on RNA binding by ITC, in the context of the longer soluble monomeric construct. An increase in KD of 9- and 6-fold was observed for NBLA-S158EE with SL1-dI and full-length linear SL1, respectively, compared to wild-type NBLA (Fig. 6D, E), indicating that S158EE significantly reduces RNA binding. Together, these observations suggest that in vivo S158 phosphorylation would reduce RNA binding without significantly disrupting Hexim autoinhibition, thereby triggering release of P-TEFb as a net result.

Both monomers in Hexim1 dimer engage in high-affinity RNA binding simultaneously
Since full-length Hexim1 is a stable homodimer, the above results with Hexim1 monomeric constructs suggest that a dimer could simultaneously bind two sites on linear SL1, one for each monomer. Indeed, EMSAs using full-length SL1 or the minimum functional 7SK (SL1-SL4ms2, that has been shown to assemble in cells with MePCE–Larp7–Hexim–P-TEFb57) show that full-length Hexim1 dimer binds first to Site 1 and Site 2. At higher Hexim1:RNA ratios, another dimer subsequently binds to Sites 4 and Site 5 (Supplementary Fig. 14F, G). These Site 4 and Site 5 are likely not primary binding sites for Hexim1 in cells, rather they may serve as docking sites during the Hexim1 searching process for Sites 1 and 2.

We next used the same short SL1 constructs described above versus full-length SL1, titrated into Hexim1 NBLA monomer versus full-length Hexim1 dimer, to determine binding stoichiometry by ITC (Fig. 7 and Supplementary Fig. 24). Systematic comparison of the N values shows that the monomeric NBLA binds as one molecule per RNA to individual Sites 1, 2, and 3 (N = 0.62–0.93) and binds as two molecules per full-length linear SL1 (N = 0.36) (Fig. 7A, B). Full-length Hexim1 (dimer) also binds as one monomer per individual Sites 1, 2, and 3 (N = 0.68–0.79, using monomer concentrations when fitting titration curves), and binds as two monomers, i.e., one dimer, per linear SL1 (N = 0.39) (Fig. 7A, C). We further verified the stoichiometry by reversing the titration order (i.e., titrated Hexim1 from the syringe to RNA in the cell), and again observed one monomer per single-site RNAs (N = 0.87–1.12) and two monomers (one dimer) for linear SL1 (N = 1.75). Together with the NMR and EMSA results, we conclude that both monomers within Hexim1 dimer are engaged in SL1 RNA binding, to Sites 1 and 2, simultaneously (Fig. 7C).

Hexim1 autoinhibition renders specificity for linear 7SK SL1
After mapping the individual RNA binding sites with their relative order of affinities and determining the stoichiometry of Hexim1 dimer binding with SL1, we next compared the ITC-determined binding affinities between NBLA and Hexim1 dimer, where inter-monomer autoinhibition is enforced by dimerization in the full-length Hexim1 but not in the monomer, to quantitatively determine the effect of Hexim1 autoinhibition on RNA binding. Comparison of the dissociation constants KD shows that the Hexim1 dimer binds 10-, 5- and 8-fold weaker than the monomer (NBLA) to the individual Site 1, Site 2, and Site 3, respectively, suggesting that autoinhibition of Hexim1 dimer weakens binding to single sites (Fig. 7). However, Hexim1 dimer binding to linear SL1 shows cooperative binding with a >10-fold lower KD (73 nM) than the single Site 1 and Site 2 (780 and 885 nM). In contrast, no cooperativity was observed for NBLA monomer, with a KD value for SL1 (336 nM) slightly larger than those of Site 1 and Site 2 (83 and 189 nM). This cooperative effect that is exclusively observed for the dimer is recapitulated in experiments where the titration order is reversed, albeit with a smaller effect likely due to increased aggregation when Hexim1 is concentrated in the syringe (Fig. 7A bottom). These results indicate that the autoinhibition of Hexim1 dimer is required for the specificity towards full-length linear 7SK SL1 over single RNA sites, whereas NBLA monomer lacks this specificity.

The weaker single sites SL1-dII (Site 4) and SL1-p (Site 5) exhibited larger KD values (1221 and 2208 nM, respectively) than Site 1 or Site 2 for Hexim1 dimer. Interestingly, the N values for these weaker binding sites are slightly smaller (0.47 and 0.49), indicating a partial release of autoinhibition such that only one monomer is available for binding within a subpopulation of the dimer. In contrast, SL1alt (Site 3) in circular 7SK conformation has weak binding affinity to Hexim1 dimer (2035 nM), albeit with a similar N value (0.68) to Sites 1 and 2 (0.76 and 0.79). This Hexim1 dimer–SL1alt affinity is unexpectedly much lower than the NBLA monomer–SL1alt, suggesting significantly different binding kinetics for dimer–Site 3 from monomer–Site 3. Consistent with these results, a SL1-d(api)27 (identical to SL1-d except for the apical loop sequence) has an enhanced binding affinity (96 nM) but a smaller N value (1.40) than SL1, indicating two binding events overall (Sites 1, 4 vs 1, 2) but with a moderately reduced release of Hexim1 autoinhibition.

Finally, to further test the role of Hexim1 autoinhibition in RNA-binding specificity, we disrupted the autoinhibition with three Hexim1 dimer variants, A3m and A12m, that neutralized negatively charged residues in c-AR and n-AR with alanines7, and ΔPYNT, and titrated them with the low-affinity binder SL1-p (Site 5) monitored by ITC (Fig. 7 and Supplementary Fig. 25). These three variants showed 8-, 6- and 5-fold tighter binding affinities than wild-type Hexim1, respectively, suggesting that disruption of autoinhibition turned Hexim1 into a non-specific RNA binder.

Together, these results show that Hexim1 dimer preferentially and cooperatively binds Sites 1 and 2 in linear SL1 with each monomer, respectively, and the inter-monomer autoinhibition within the dimer renders the specificity for linear SL1 over isolated RNA binding sites, including the circular SL1alt. This is achieved through two sides of the same coin: cooperative binding to the dual high-affinity sites in SL1 and decreased affinity for RNAs with only a single binding site. Together with the PRE results, we conclude that Hexim1 binding to two sites in linear SL1 provides an effective allosteric switch to fully release Hexim1 autoinhibition in the dimer, which subsequently exposes two PYNT motifs for Cdk9 binding and inactivation from two P-TEFb heterodimers (Fig. 8).

Methods
Plasmids and mutagenesis
Full-length human Hexim1 protein (aa 1–359) and monomeric Hexim1 BR-L-AR (aa 142–253) were cloned into pETDuet-1 vector with an N-terminal His6-tagged maltose binding protein (MBP) fusion, followed by Tobacco Etch Virus (TEV) protease cleavage site between MBP and Hexim1. Further truncated monomeric Hexim1 constructs, BR-L (aa 136–209) and BRext (aa 149–179) were cloned into pETDuet-1 vector with N-terminal His6-tagged 56-residue B1 domain of Streptococcal protein G (GB1) fusion70 followed by Tobacco Etch Virus (TEV) protease cleavage site between GB1 and Hexim1 monomers. BR (aa 141–165) and BRARM1 (aa 141–159) constructs were cloned by site-directed mutagenesis using the pETDuet-1 GB1-ts-BR-L plasmid as template. A non-native glycine remains at the N-terminus of the final full-length Hexim1 protein, BR-L-AR, BR-L, BR and BRARM1 after TEV protease cleavage, whereas a native Glycine residue from BRext (aa 149) was a part of the TEV cleavage site (ts). Cysteine substitutions of BR-L-AR variants for PRE (S183C, A201C, S226C, S233C and S237C) were cloned by site-directed mutagenesis using the pETDuet1 MBP-ts-BR-L-AR as template.

Minimum linear 7SK (SL1-SL4ms2) and linear SL1 RNA genes were cloned into the pUC19 vector with a Hammerhead ribozyme gene at the 3′ end followed by a BamHI restriction enzyme cleavage sequence.

All site-directed mutagenesis was performed using the Q5 kit (New England Biolabs).

Protein expression and purification
Hexim1 protein genes in either pETDuet-MBP or pETDuet-GB1 vectors were transformed into BL21-GOLD (DE3) competent cells for protein expression. Bacterial culture was grown at 37 °C in minimum media to O.D.600 of 0.6, then transferred to 18 °C for 1 hr prior to induction by 0.5 mM IPTG for 18–20 hrs. For uniformly [15N, 13C] (or [15N]) enriched Hexim1 proteins, M9 minimal media containing 1 g/L of 15NH4Cl and 3 g/L of [13C-6]-D-glucose (or natural abundance glucose) (Cambridge Isotope Laboratories) were used. Cells were pelleted by centrifugation, resuspended in lysis buffer (20 mM Tris, pH 7.5, 1 M NaCl, 5 mM imidazole and 1 mM PMSF) supplemented with protease inhibitor cocktail tablet (Thermo Scientific Pierce A32965) and lysozyme. Cells were lysed by sonication on ice and clarified by centrifugation and filtration with 0.45 μm syringe filter. Lysates were loaded onto a 5-mL Ni Sepharose affinity column (HisTrap-HP; GE Healthcare), and a linear gradient of 5–500 mM imidazole was used to elute the His6-tagged proteins. The eluted protein was added 1 mg of TEV protease, dialyzed (20 mM Tris, pH 7.5, 300 mM NaCl, 5 mM βME) and further purified with a second Ni affinity column to remove His6-tagged fusion proteins (MBP or GB1) and TEV protease. For the full-length Hexim1 protein, an additional purification step was carried out with size-exclusion chromatography (SEC; HiLoad 26/600 Superdex 200 or HiLoad 16/600 Superdex 75; GE Healthcare) in binding buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 1 mM TCEP). Proteins were concentrated using Amicon devices (Millipore), and concentration was measured by absorbance at 280 nm. For monomeric Hexim1 BR-L-AR, concentrating and buffer exchanging using Amicon devices into a final buffer (50 mM sodium phosphate, pH 6.2, 0/150/300 mM KCl, 1 mM TCEP, 0.02% NaN3, 5% D2O added post buffer exchange for NMR) was performed with caution to prevent precipitation at higher local protein concentration. For even shorter monomeric proteins, 1 kDa MWCO dialysis tubing (for BRext) or desalting column (HiTrap Desalting 5 mL, GE Healthcare; for BR and BRARM1) were used for buffer exchange into 5 mM NH4HCO3 buffer, freeze dried with a lyophilizer and dissolved directly in NMR buffer (50 mM sodium phosphate, pH 6.2, 150 mM KCl, 0.02% NaN3, 5% D2O).

In vitro phosphorylation by PKA catalytic subunit
Phosphorylation of Hexim1 BR-L-AR was performed utilizing cAMP-dependent protein kinase A catalytic subunit (PKAcat; NEB P6000S). The protein was first buffer-exchanged into NMR buffer (50 mM sodium phosphate, pH 6.2, 150 mM KCl, 0.02% NaN3, 5% D2O), then concentrated with constant mixing using Amicon devices (Millipore). The final phosphorylation reaction (500 μL total volume) contained 35 μM BR-L-AR, 2 mM ATP, 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, and 4 μL PKA (10,000 units). Two separate reactions were incubated in a water bath at 37 °C for 3 and 24 h, respectively, before being analyzed using NMR.

Native PAGE and mass photometry characterization of Hexim1 constructs
Prior to loading, freshly purified protein samples of full-length Hexim1, L-AR-CC, and BR-L-AR were centrifuged at 14,000 × g for 10 minutes at 4 °C to remove potential contaminants and aggregates. Samples were prepared with Native PAGE™ Sample Buffer (4X) (Invitrogen BN2003). For full-length Hexim1 and L-AR-CC, 5 μg was loaded per lane. For BR-L-AR, 2.5 μg and 5 μg were loaded on separate lanes. Samples were run on Native PAGE™ 3–12% Bis-Tris Mini Gels (Invitrogen™ BN1003BOX) at room temperature at 150 V using a cathode buffer supplemented with Coomassie Blue G-250 at a final concentration of 0.02% (Invitrogen™ BN2007).

Mass photometry measurements of full-length Hexim1, L-AR-CC, and BR-L-AR were performed on a Refeyn TwoMP mass photometer. Freshly purified protein samples were prepared as 4 μM for monomers and 2 μM for dimers, diluted (or mixed as complexes and diluted) with SEC buffer to 400 nM stocks, and then added by the drop-dilution method into phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 pH 7.4) to achieve final total protein concentrations of 40 nM. Interferograms were recorded for 60 seconds with Refeyn AcquireMP, and data were analyzed with Refeyn DiscoverMP using three molecular weight standards for calibration: Bovine Serum Albumin (BSA, ThermoFisher 23209) 66 kDa/132 kDa, beta-amylase (BAM, Sigma A8781) 112 kDa/224 kDa and Bovine Thyroglobulin (TG, Sigma 609310) 670 kDa (R2=1.000, Max. mass error 3.0%). The percentages associated with each peak fitting are derived from the DiscoverMP analysis, which calculates the proportion of total particle counts represented by the Gaussian-fitted peaks.

Nitroxide spin labeling of proteins
Protein modification with nitroxide spin label was carried out using the established protocol71. Briefly, each purified protein solution, containing about 50 nmole of Hexim1 BR-L-AR cysteine variants, was buffer exchanged using a PD-10 desalting column with the gravity protocol to remove the reducing agent into PRE buffer (50 mM sodium phosphate, pH 6.2, 150 mM KCl, 0.02% NaN3). The PD-10 elution was collected into a falcon tube, containing 10-fold molar excess of S-(1-oxyl-2,2,5,5,-tetramethyl-2,5,-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothiolate (MTSL; Toronto Research Chemicals TRC-O875000) dissolved in 0.5 mL of PRE-buffer, covered with aluminum foil to avoid light. The reaction was kept at room temperature under gentle stirring in the dark for >15 min. Subsequently, an additional aliquot of MTSL was added to the reaction mixture to reach a final ratio of 1:20 protein:MTSL. The reaction was further incubated at room temperature under gentle stirring overnight in the dark.

The unreacted MTSL molecule was removed by running through a PD-10 desalting column equilibrated in PRE buffer twice using the gravity protocol. The MTSL-tagged protein was concentrated with caution using Amicon devices, and 5% v/v D2O was added to prepare the final NMR sample.

In vitro transcription of RNA
RNA samples were prepared by in vitro transcription with T7 RNA polymerase P266L variant34,72 and are described briefly below. Short RNA constructs, including linear 7SK SL1 dissected constructs SL1-dI, SL1-dIΔU63, SL1-dII, SL1-d, SL1-dΔU63 SL1-dIIm, SL1-m, SL1-mp, SL1-pII, SL1-pIIo, SL1-p, and circular 7SK SL1alt (wt or UUCG apical loop), were transcribed from chemically synthesized DNA oligonucleotides (Integrated DNA Technologies). Transcription mixtures (40 mM Tris, pH 8.0, 1 mM spermidine, 0.01% Triton-X100, 2.5 mM DTT, 25 mM MgCl2, 0.5 μM oligonucleotide DNA template, T7 RNAP P266L, 4 mM each rATP, rUTP, rGTP and rCTP) were incubated at 37 °C for 4–6 hrs. Longer RNA constructs, minimum linear 7SK (SL1-SL4ms2) and linear 7SK SL1, were transcribed from plasmid DNA templates prepared with QIAGEN Plasmid Maxi Kit and linearized with BamHI-HF restriction enzyme (New England Biolabs). Transcription mixtures are the same as the short RNA constructs above, except from lower MgCl2 concentration (15-20 mM) and overnight incubation at 37 °C.

Transcription mixtures were loaded onto 10–20% denaturing polyacrylamide gel electrophoresis (Urea-PAGE; 19:1 crosslinking ratio) for RNA purification. Purified RNA molecules were eluted out of gel pieces either with an Elutrap device (GE Whatman) or by crush-and-soak in 1X TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.3). Collected RNA eluents were concentrated and buffer exchanged into sterilized nanopure water using 3 kDa or 10 kDa MWCO Amicon devices (Millipore), supplied with counterion in high salt buffer (10 mM sodium phosphate, pH 7.6, 1 mM EDTA, 1.5 M KCl) and buffer exchanged back into sterilized nanopure water. Dilute RNA samples <100 μM were heated at 95 °C for 5 min and snap cooled on ice for 1 hr prior to final buffer exchanging and concentrating with Amicon devices (Millipore). Stock RNA solutions were exchanged into sterilized nanopure water and stored in −20 °C freezer.

Electrophoretic mobility shift assay
1 μL of 50 μM RNA and stock solutions of 25–50 μM Hexim1 proteins were used to achieve varying protein-to-RNA molar ratios in a total volume of 5.5 μL in binding buffer, and added 1 μL loading dye (30% glycerol and bromophenol blue). The binding mixtures were incubated at room temperature for 15 min prior to gel electrophoresis on 5.7% non-denaturing polyacrylamide gel (PAGE; 37.5:1 crosslinking ratio) or 0.8% Agarose gel (for minimum linear 7SK SL1-SL4ms2) in 0.5X TBE buffer at room temperature. Polyacrylamide gels (BioRad Mini-PROTEAN hand cast) and agarose gel (BioRad Mini-Sub 7×7 cm) were run at 70 V. Polyacrylamide gels were subsequently stained with Toluidine blue solution and destained in water, whereas agarose gels were made with SYBR™ Safe stain (Invitrogen) added prior to solidifying. Gels were imaged with a Pharos FX Plus scanner (Bio-Rad), and band intensities were extracted using ImageJ and fit to binding models (see Supplementary Fig. 14 notes) with Igor-Pro 9 software.

NMR spectroscopy
For NMR assignments of SL1-dIΔU63 RNA bound BR-L-AR, dilute BR-L-AR uniformly enriched with 15N and 13C were combined with dIΔU63 in a 1:1.2 molar ratio, and the dilute complex was buffer exchanged in NMR buffer (50 mM sodium phosphate, pH 6.2, 150 mM KCl, 0.02% NaN3, 5% D2O) and concentrated down to a final concentration of 0.22 mM BR-L-AR and 0.25 mM SL1-dIΔU63. Backbone assignments were carried out using the following NMR experiments: HNCACB, CBCA(CO)NH, HNCA and C(CO)NH. For NMR assignments of Hexim1 BR-L-AR, 70 μM sample uniformly enriched with 15N and 13C in the NMR buffer was used. Backbone assignments were carried out using the CBCA(CO)NH and HNCA NMR experiments, which were collected with non-uniform sampling and processed with iterative soft thresholding reconstruction73, and comparison to the RNA-bound BR-L-AR assignments.

For NMR assignments of further truncated Hexim1 constructs, BR-L, BR, BRext and BRARM1, samples uniformly enriched with 15N and 13C in the range of 0.5-0.8 mM concentration in NMR buffer were used. Backbone assignments were carried out using the following NMR experiments: HNCACB, CBCA(CO)NH and C(CO)NH. Additional side-chain assignments were acquired for BR-L and BR using HCCH-TOCSY. BRARM1 15N-1H HSQC resonances were assigned based on almost complete overlapping with the BR 15N-1H HSQC peaks. For 1H imino sequential assignments of RNA samples in the absence and presence of monomeric Hexim1 proteins, the two-dimensional NOESY spectra were recorded using 5% D2O samples at 283.15 K with NOE mixing time of 150 ms. For PRE NMR experiments, 15N-1H HSQC spectra were recorded at 290.15 K with Avance III HD 600 MHz.

For NMR assignments of 7SK RNA constructs, natural abundance samples in the range of 0.15–1.24 mM concentration in NMR buffer (50 mM sodium phosphate, pH 6.2, 150 mM KCl, 0.02% NaN3, 5% D2O, with the exception of 0 mM KCl NMR buffer for SL1-dIΔU63, SL1-dII and SL1-d, and 10 mM sodium phosphate, 50 mM KCl, pH 6.3, 5% D2O for SL1altUUCG) were used. For secondary structure determination and N-H…N imino proton assignments (exchangeable-proton, here H1 for Gua and H3 for Ura), NOESY and TOCSY spectra were recorded using 95% H2O/5% D2O samples at both 283 K and 298 K. For non exchangeable proton assignments (here H2/H8 for Ade, H8 for Gua, H5/H6 for Cyt/Ura, and H1′ for all nucleotides), NOESY and TOCSY spectra were recorded using 100% D2O samples at both 283 K and 298 K. The 13C chemical shifts from the non exchangeable C-H vector were assigned by mapping proton assignments on 13C-1H HSQC spectra individually recorded for the aromatic region (C8-H8, C6-H6 and C2-H2) and for the remaining vectors (C1′-H1′ and C5-H5). Protein-RNA binding experiments were all performed in NMR buffer of 50 mM sodium phosphate, pH 6.2, 150 mM KCl, 0.02% NaN3, 5% D2O.

For the chemical shift perturbation (CSP) analysis of RNA constructs, observed imino chemical shift values (H3 from Us and H1 from Gs) together with H2 of Ade from A-U base pairs were used to calculate the change in chemical shift Δ between the free and protein-bound states. For Site 1, Site 4 and Site 5 constructs, additionally assigned nonexchangeable protons were included in the CSP analyses. Protein-bound RNA spectra of NOESY and TOCSY were assigned by tracing peak trajectories in the corresponding titration series, and only assignments of high confidence were included in the final analyses. Severely broadened and highly overlapping residues were excluded from the CSP calculations.

For the CSP analysis for Hexim1 proteins, the overall change in chemical shift Δ were calculated for protein 15N-1H HSQC resonances between the free and RNA-bound states using the following equation:
Δ = sqrt(ΔδH^2 + 0.152 × ΔδN^2)
where ΔδH and ΔδN are the differences between the 1H and 15N chemical shifts of the two states being compared.

All the NMR spectra were collected at 298 K with an Avance Neo 800 MHz spectrometer equipped with TCI H&F cryoprobe, an Avance III HD 600 MHz equipped with QCI HCNP cryoprobe or a Bruker DRX 500 MHz with cryoprobe. Data were collected with TopSpin (Bruker), processed with NMRPipe74, and analyzed in NMRFAM-Sparky75. Hexim1 protein CSP values and PRE intensity ratios were plotted and visualized with Igor-Pro 9 software.

Isothermal titration calorimetry
The dissociation constants (KD) for binding between Hexim1 proteins and 7SK RNA constructs were determined using a MicroCal 200 ITC instrument (GE). RNA and protein were buffer exchanged using separate 3 kDa MWCO dialysis tubings into the same bucket of ITC buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 1 mM TCEP). Dilute dialyzed RNA solutions <20 μM were heated at 95 °C for 5 min and snap cooled on ice for 1 hr right before the first set of titration experiments. RNA final concentrations were determined by UV absorbance at 90 °C at 260 nm with the Lambert-Beer law, using a Hewlett-Packard HP8453 diode-array UV/Visible spectrophotometer with Peltier temperature control. Extinction coefficients of melt RNA were calculated by the Oligo Analyzer Tool from Integrated DNA Technologies. RNA stock solutions were kept at room temperature to avoid cold-induced oligomerization for the 2-3 days duration of one complete titration set. RNA at concentration 63-195 μM in the syringe was titrated into 7–40 μM protein in the sample cell at 295 K, or for reversed titration order protein at concentrations of 102–120 μM in the syringe was titrated into 6–12 μM RNA in the sample cell at 295 K. Calorimetric data was fit using ORIGIN 7 (Micro-Cal). The binding parameters stoichiometry (N), entropy (ΔS), enthalpy (ΔH) and association constant (KA) were kept as floating variables during each fit. ITC experiments were performed in biological triplicate or quadruplicate (n = 3 or 4) to verify reproducibility and estimate experimental error. No statistical methods were used to predetermine sample sizes, as n = 3 is standard for quantitative biophysical measurements of purified systems. Full thermodynamics parameters and individual replicate numbers are listed in Supplementary Table 1.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Yang et al. describes a detailed NMR mapping and conformation analysis of the human protein Hexim1 targeting the small nuclear RNA 7SK. The 5’ stem loop (SL1) of the 332 nt 7SK RNA interacts with its bulge regions with the basic regions of the homodimer Hexim1. Two different conformations have been proposed for 7SK SL1 (linear and circular) with different conformations and accessibilities of GAUC-containing bulge regions. Using sophisticated NMR-mapping experiments of a central construct of Hexim1, which is composed of a basic region, a linker region, and an acidic region (BR-L-AR, together 112 aa) or parts of it (BR-half, BR, BR-L), in combination with different constructs of the 7SK RNA, the authors identify two high affinity binding sites (1 and 2) and two lower affinity binding sites (4 and 5) in the linear conformation of SL1 as well as one high affinity binding site (3) in the circular conformation of SL1. The interactions to the three high affinity binding sites are validated by ITC experiments (Kds of 83 to 230 nM). Using the cysteine-reactive paramagnetic spin label MTSL in combination with site-directed mutagenesis for covalent attachment, the authors identify sites of extensive line broadening that give rise to conformational changes. They propose an autoinhibited masking of the central CDK9-inhibition motif PYNT by the flaking basic and acidic regions that might act in trans in the Hexim dimer assembly (Fig. 6).\nOverall, this a sound NMR study of a conformation and interaction analysis of Hexim1 with 7SK SL1 RNA. The manuscript is hard to follow for a non-NMR-specialist (particularly pages 9-11), making it a weaker candidate to the transcription regulation-interested broader community. Text and Figures, however, are well prepared. I have only a few remarks to this detailed and specific study.\nComments:\nSer158 in the basic region of Hexim1 has been described to be phosphorylated to regulate P-TEFb activity. The authors should characterize a BR-L-AR mutant where S158 is mutated to E (or even EE) to analyse conformational changes of the autoinhibited structure with a phosphorylation mimicry.\nThe C-terminal Cyclin T1/T2 coiled-coil binding domain (shown e.g. in the cartoon display of Fig. 1) has been determine by NMR spectroscopy including its chemical shift assignment. The N-terminal extension of this construct was shown to be feasible.\nThe linker region has been well analysed in previous studies (PMID 22588304) including a detailed characterization of the inhibitory potential of the PYNT motif in kinase activity experiments, showing that T205 (but not Y as e.g. in p27-Kip1 for CDK2 inhibition) is the main driver of kinase inhibition. The statement that “The central part of Hexim1 (150-250) has not been characterized previously, with the exception of a small region (~20 aa nuclear localization signal stretch) that binds 7SK RNA, also referred to as Basic Region (BR, aa 150-169)7,8.” (Introduction section, page 3) seems therefore not correct giving previous work described in references 9 and 19.", "reviewer_2": "The 7SK RNA-protein complex (RNP) is an important factor for transcription regulation because it sequesters P-TEFb the kinase complex (CDK9 and Cyclin T1) that is required for transcription elongation after RNA polymerase II pausing. There were several 7SK states previously detected in cells but for P-TEFb regulation a core complex consisting of 7SK RNA, HEXIM1 (or 2), MePCE and LARP7 seems to be crucial. Here, Yang, Feigon and colleagues try to define how HEXIM1 contacts 7SK RNA and how RNA binding influences its conformation. The authors generated a large number of truncated 7SK RNA sections and several HEXIM1 truncation constructs which they use for NMR and binding assays (EMSA, ITC).\nThe paper is terribly confusing and very hard to read. Some of this is due to different sets of protein constructs and RNA designs being used for different assays, but also the text jumps back and forth between figures, does not explain the relevance of NMR findings in general terms, and for several RNA constructs it is difficult to find out what they correspond to. It is often unclear, what conclusions are based on because the presented data does not warrant the conclusions.\nIt is most problematic, that some assays (NMR, EMSA, ITC) were carried out with one set of RNA and protein construct, while others were only done with different sets. But then conclusions from the first set are simply \"carried over\" to the second set of constructs. Furthermore, quite ambivalent PRE data is overinterpreted to fit into an autoinhibition model. In its current form, I do not see how the conclusions that were made are supported by experimental evidence. To change this issue, minimally EMSA and ITC binding assays as well as NMR titration experiments would have to be carried out for corresponding and meaningful sets of protein/RNA constructs. Also complementary evidence to the current PRE data is required to make the autoinhibition model convincing.\nIn addition, the artificial nature of the shortened RNA and protein constructs, as well as the minimal set of relevant components used in the assays (only part of 7SK RNA and HEXIM1) makes it unclear how relevant the study is to understand what happens in cells and what any of the findings tell us of how 7SK-HEXIM1 binding to P-TEFb could be regulated.\nIn summary, before publication can be considered this manuscript needs severe restructuring and streamlining of text and figures, plus significant experimental work.\nBelow, I am listing some of the issues in detail in the hopes that it helps to improve the manuscript.\nRNA and protein constructs:\n* It is often unclear what the RNA constructs used in each experiment exactly look like, these should be shown as inserts in each assay or minimally the complete set should be shown in Fig.3A.\n* Why are deltaU constructs used? From the EMSA studies it seems that SL1-dIdeltaU binds much less well to HEXIM1 than SL1-dI (compare Suppl.Fig.5B gel1 and gel2). Yet, in ITC experiments only SL1-dI is used and not the corresponding deltaU RNA. But then deltaU constructs are used for NMR. These are completely artificial constructs, why are they relevant?\n*Why are different sets or RNAs used for EMSA vs. ITC vs. NMR? Why are different HEXIM1 constructs used for EMSA vs. ITC vs. NMR experiments? I understand that solubility is an issue for some protein constructs, which makes it difficult to use them for NMR but for ITC and EMSA this should not be a problem, given that RNA affinities are in the nano to micro molar range. These differences make the manuscript not only confusing and difficult to follow, they also draw comparisons and conclusions into question.\nRNA NMR experiments and CSPs:\n*p.8 states \"Using chemical shift perturbation (CSP) of RNA imino proton resonances in 1H-1H NOESY spectra in the absence versus presence of monomeric Hexim1 BR-L-AR or BR, we identified SL1 Sites 1 and 2 as two high affinity binding sites, and Sites 4 and 5 as two lower affinity binding sites (Fig. 3 and Supplementary Fig. 7).\"\n-> Several problems here: # How can CSPs inform on high/low affinity binding sites?\n# CSPs for RNA is never shown anywhere, only (difficult to distiguish) shades of blue in the base letters (Fig.3A, S5A) indicate them to some extent. CSPs should be shown in a graph analogous to protein CSPs.\n# NMR experiments are done with inconsistent protein constructs, which on top differ from what the sentence stated i.e. SL-dIdelaU with BR-L-AR, SL1-dII with BR-L, SL1-mdII with BRhalf. This means, BR is never used, instead BR-L and BRhalf were used but only for one RNA each. If both protein and RNA are changed in each experiment, how can conclusions about affinities to individual RNA binding sites be made?\n*p.8 states \"when using longer 7SK RNA constructs that include two adjacent binding sites, the higher affinity site of the two is occupied by stoichiometric addition of Hexim1 BR as shown by NMR CSP of RNA iminos, i.e. in SL-d Site 1 shows CSP instead of Site 4, in SL1-mdII Site 2 shows CSP instead of Site 4, and in SL1-pm Site 2 shows CSP instead of Site 5 (Fig. 3A and Supplementary Fig. 5A);\"\n-> Where is this data? There is no SL1-d NMR dataset shown anywhere. Furthermore, binding site 4 in SL1-d in the scheme is shown in grey, which means \"not assigned\". So how can any conclusion about protein binding to this region be made?\nEMSAs/ITC - Several issues with presentation, analysis and interpretation of EMSAs and ITC experiments:\n*Instead of reporting RNA-protein ratios, the EMSA binding assays should rather report concentrations. Kd values should be calculated and compared to ITC data.\n*It is unclear how the assignments on the side of the EMSA gels with binding sites 1/2/4/5 can be made, especially for SL1-ddeltaU, when both site 4 and site 1 were bound separately at roughly 0.9 RNA/protein ratio (compare Suppl.Fig.5B gel1 and gel3). All that can be concluded is that the first shift has probably 1 HEXIM1 bound and the second shift has probably 2 HEXIM1s bound to the RNA.\n*Similarly, statements in the text are not supported by the data. E.g. p.9 \"Indeed, EMSAs of SL1 constructs with full-length Hexim1 show that Hexim1 dimer binds first to Sites 1 and 2 and subsequently to Sites 4 and 5 (Supplementary Fig. 5D,E).\"\nAll that can be concluded from this EMSA is that two proteins bind at 1.2 ratio and another two at 2.4 ratio. Which RNA sites they bind to cannot be seen in this assay.\n*All ITC curves should be included in the supplement. Some of the listed affinities are comparably weak (micro molar Kd) and fitting gives substoichiometry N-values, which could indicate that these curves do not reliably report N or Kd any more and should rather be given as approximate boundary values (rather than four significant digits).\nPRE experiments and PYNT autoinhibition model:\n* In Fig. 4B-E regions with PRE intensity ratios below mean-0.5sigma and mean-1sigma are supposedly shaded in orange and red. Yet in almost every one of the shaded regions several residues lie above the supposed cut-off (or are simply not assigned) and conversely in the white regions several residues have lower than cut-off ratios (or are simply not assigned). Thus, the shaded areas give an oversimplified and scewed picture of the data and this is cemented further by the structure cartoons presented on top. Based on the data one could equally well argue that the AR is in transient contact with any one of the other regions of HEXIM1. Additional and complementary evidence would be required to make this section convincing.\n* On top of the weak data, for these experiments it is especially relevant that they are carried out in the context of a truncated HEXIM1 that cannot form the obligate dimer. The N-terminal HEXIM1 regions could equally well bind back to the coiled-coil section, which would also lead to reduced RNA affinity of full length HEXIM1 for 7SK RNA (as determined by ITC).\n* If the PYNT motif is truely involved in reducing RNA affinity and HEXIM1 making binding \"more selective\", then a deletion of this short motif should increase RNA affinity of HEXIM1 and make it \"less selective\".\n* Conceptually the model also poses a problem as most of HEXIM1 in cells is bound to dsRNA (Li et al. NAR 2007), and should therefore be relieved of the autoinhibited conformation and associate with P-TEFb according to the proposed model, but this seems not to be the case in cells. Furthermore, in previous studies the very N-terminus that is not studied here (aa 1-120) has an auto-inhibitory effect (Li et al. JBC 2005).\nFigure citations - The text jumps back and forth between figures some in the wrong order and some figure citations seem to be entirely wrong e.g. Fig. 2B should be cited on p.5 (\"...referred to as BR-L-AR in the following text.\") before Fig. 2A is coming in. There is no Fig.3E but it is referred to in the text (p.7 \"...bulge (U40U41) is also present 3’ to the alternative GAUC palindrome (Fig. 3E).\")\nChemical exchange - If this paper is meant for a general audience, the authors should explain what slow, intermediate and fast exchange mean in context of the system studied and the complexes that are formed here (e.g. on p.6, p.8).\nAbstract final sentence \"These results provide mechanistic insights into Hexim–RNA specificity and explain how P-TEFb can be effectively regulated to respond to changing levels of transcriptional signaling.\" - This conclusion is not warranted given that the authors have not carried out any assays in cells and used only artificial in vitro systems with few (and mostly severely truncated) components.\nFigure inconsistencies - I understand that some peaks are broadened upon addition of RNA but it still looks like some bars are missing in Suppl.Fig.4C basic region in comparison to Fig. 2D/E? Also the baseline at 0 is missing in Suppl.Fig.4C.\nSuppl. Fig. 1 and 2: The sequence alignments seem to be cut out of larger alignments because they display numerous empty columns that hold no information. These empty columns should be removed to make the alignments more compact and easier to understand."}, "author_response": "We thank the reviewer for the positive comments and the concern for the suboptimal readability towards non-NMR specialist readers. To address this important concern, we have significantly rewritten and rearranged the manuscript, including the NMR sections, and incorporated new and enlarged cartoons to help with visual interpretation of the NMR results (revised Figs. 3 and 4). We also now have a new section on S158 phosphorylation and substitution that should increase the interest of the broader transcription community.\nComments:\nSer158 in the basic region of Hexim1 has been described to be phosphorylated to regulate P-TEFb activity. The\nauthors should characterize a BR-L-AR mutant where S158 is mutated to E (or even EE) to analyse conformational\nchanges of the autoinhibited structure with a phosphorylation mimicry.\nResponse:\nWe thank the reviewer for this great suggestion. To this end, we performed two additional sets of experiments: (1) in\nvitro phosphorylation of BR-L-AR by PKA catalytic subunit, which showed S158 phosphorylation (assayed by NMR)\nhad minimal impact on the autoinhibited conformation, and (2) ITC binding measurements of a NBLA-S158EE mutant\nbinding to 7SK, which showed significantly reduced RNA binding. Together, these observations suggest that in vivo\nS158 phosphorylation leads to reduced RNA binding without significantly disrupting Hexim autoinhibition, thereby\ntriggering release of P-TEFb as a net result. See the new Results section titled “S158 phosphorylation minimally\naffects autoinhibition and S158EE impairs RNA binding” (Lines 289–312), new Fig. 6 and Supplementary Fig. 23, of\nthe revised manuscript, for details.\nThe C-terminal Cyclin T1/T2 coiled-coil binding domain (shown e.g. in the cartoon display of Fig. 1) has been\ndetermine by NMR spectroscopy including its chemical shift assignment. The N-terminal extension of this construct\nwas shown to be feasible.\nResponse:\nWe were aware of the excellent papers on the Hexim coiled-coil domain from the Geyer lab, and apologize for\nneglecting to reference the NMR structure in our previous manuscript. Regarding N-terminal extension of coiled-coil\nconstruct, we could indeed express and purify soluble proteins of BR-L-AR-CC and L-AR-CC constructs.\nUnfortunately, BR-L-AR-CC construct exhibited severe line broadening for the entire BR-L-AR region by NMR, similar\nto full-length Hexim1, despite our efforts in optimizing temperature, salt, and deuterium labeling of the constructs. We\ntherefore included new nativePAGE and mass photometry data including Hexim1 dimer and L-AR-CC dimer in our\nrevised manuscript (Lines 109–126, Fig. 1 and Supplementary Fig. 4), which provide strong evidence of stable inter-\nmonomer interactions.\nThe linker region has been well analysed in previous studies (PMID 22588304) including a detailed characterization\nof the inhibitory potential of the PYNT motif in kinase activity experiments, showing that T205 (but not Y as e.g. in\np27-Kip1 for CDK2 inhibition) is the main driver of kinase inhibition. The statement that “The central part of Hexim1 (150-250) has not been characterized previously, with the exception of a small region (~20 aa nuclear localization signal stretch) that binds 7SK RNA, also referred to as Basic Region (BR, aa 150-169)7,8.” (Introduction section, page 3) seems therefore not correct giving previous work described in references 9 and 19.\nResponse:\nWe thank the reviewer for this correction. We meant “has not been structurally characterized”. We have clarified this\nin the revised manuscript. We have also incorporated the important details related to linker region analysis in PMID\n22588304 in the revised manuscript’s Introduction section (Lines 62).\nReviewer #2 (Remarks to the Author):\nThe 7SK RNA-protein complex (RNP) is an important factor for transcription regulation because it sequesters P-TEFb\nthe kinase complex (CDK9 and Cyclin T1) that is required for transcription elongation after RNA polymerase II\npausing. There were several 7SK states previously detected in cells but for P-TEFb regulation a core complex\nconsisting of 7SK RNA, HEXIM1 (or 2), MePCE and LARP7 seems to be crucial. Here, Yang, Feigon and colleagues\ntry to define how HEXIM1 contacts 7SK RNA and how RNA binding influences its conformation. The authors\ngenerated a large number of truncated 7SK RNA sections and several HEXIM1 truncation constructs which they use\nfor NMR and binding assays (EMSA, ITC).\nThe paper is terribly confusing and very hard to read. Some of this is due to different sets of protein constructs and\nRNA designs being used for different assays, but also the text jumps back and forth between figures, does not\nexplain the relevance of NMR findings in general terms, and for several RNA constructs it is difficult to find out what\nthey correspond to. It is often unclear, what conclusions are based on because the presented data does not warrant\nthe conclusions.\nResponse:\nWe thank the reviewer for pointing out the structural issues with our manuscript, as we had indeed a large body of\ninter-connected datasets to describe. We have significantly re-organized the Results sections, regrouped relevant\nprotein and RNA construct designs in the main and supplementary figures, and now clearly lay out what was learned\nat the end of each section. We hope this along with additional new experiments will convince the reviewer that our\ndata does indeed warrant our conclusions.\nIt is most problematic, that some assays (NMR, EMSA, ITC) were carried out with one set of RNA and protein\nconstruct, while others were only done with different sets. But then conclusions from the first set are simply \"carried\nover\" to the second set of constructs. Furthermore, quite ambivalent PRE data is overinterpreted to fit into an\nautoinhibition model. In its current form, I do not see how the conclusions that were made are supported by\nexperimental evidence. To change this issue, minimally EMSA and ITC binding assays as well as NMR titration\nexperiments would have to be carried out for corresponding and meaningful sets of protein/RNA constructs. Also\ncomplementary evidence to the current PRE data is required to make the autoinhibition model convincing.\nResponse:\nWe have rewritten the Results to address the confusions regarding different constructs, and added comparisons\nbetween constructs whenever possible. In addition, we have now included nativePAGE and mass photometry results\nto provide complementary analysis for the PRE results, and acquired new evidence for inter-monomer interactions\nmediated by the central region for both NMR-range micromolar concentration (nativePAGE) and dilute nM\nconcentration (mass photometry). These results have been incorporated into the revised manuscript (Lines 109–126,\nFig. 1 and Supplementary Fig. 4).\nIn addition, the artificial nature of the shortened RNA and protein constructs, as well as the minimal set of relevant\ncomponents used in the assays (only part of 7SK RNA and HEXIM1) makes it unclear how relevant the study is to\nunderstand what happens in cells and what any of the findings tell us of how 7SK-HEXIM1 binding to P-TEFb could\nbe regulated.\nResponse:\nFirst, we already know that the two 7SK RNA conformations and a minimum Site1 were relevant both in vitro and in\ncells from the body of literature, especially from our recent structure/function work (Yang et al. Mol Cell 2022) and\nDANCE-MaP ensemble-deconvoluted chemical probing work (Olson et al. Mol Cell 2022), which both illustrated the\ncorrelation between 7SK RNA conformations and Hexim1/P-TEFb sequestration/release in cells. Thus, studying the\nsite-specific difference between linear and circular 7SK in binding Hexim is key to understanding the eukaryotic\ntranscription regulation by 7SK RNP.\nSecond, it is standard to use divide-and-conquer strategy to dissect RNA binding sites and compare to larger RNA\nand protein, since many RNA (and DNA)-binding proteins exhibit both specific and non-specific binding modes. Since\nprevious structural and functional studies on Hexim–7SK binding focused exclusively on linear 7SK Site1 and on\nBRARM1 or BR of Hexim1, and the fact that Hexim was proposed as a non-specific dsRNA binder in one study, our\nstudy here is the first that maps the complete picture of the Hexim–7SK RNA interaction landscape. Using a divide-\nand-conquer strategy to compare short to long RNA, and compare binding of Hexim1 monomer constructs [BRARM1,\nBR, BR-L, BR-L-AR, NBLA] to full-length Hexim1 dimer, was key to distinguish specific (high-affinity) versus non-\nspecific (low-affinity) RNA binding sites by monomeric Hexim1 constructs and to compare them to binding by the\nautoinhibited full-length Hexim1 dimer. We could not have understood or explained how binding of full-length Hexim1\nto dual sites on 7SK SL1 regulated autoinhibition and therefore P-TEFb binding and inactivation without these\nexperiments.\nWe also note that a previous study looked at HIV-1 Tat binding to 7SK SL4 on the basis of which it was concluded\nthat Hexim1 also binds SL4 (Durney et al. JMB 2010 PMID: 20816986). However, we now know from our structural\nand functional work (Yang et al. Mol Cell 2022) that SL4 is unavailable for Hexim1 binding in the correctly assembled\n7SK core RNP, since its major groove is fully occupied by Larp7–SL4 interaction. This highlights the need to look at\nthe physiologically relevant Hexim1 binding site on 7SK RNA, in order to avoid arriving at the wrong conclusion about\nHexim1 binding specificity, 7SK RNP assembly, or Hexim–Tat competition for 7SK. In fact, we can now confidently\nsay that SL4 (even in the absence of Larp7) is not a specific site for Hexim1 based on our current study comparing\nRNA binding sites. SL4 contains a dynamic C…C-G base triple from a CU bulge at the 3’ side of the proximal stem,\nfailing to meet the two requirements we determined for Hexim1 high-affinity binding sites: U…A-U base triple from a\nU-rich bulge at the 5’ side of the A-U rich stem.\nIn summary, before publication can be considered this manuscript needs severe restructuring and streamlining of text\nand figures, plus significant experimental work.\nBelow, I am listing some of the issues in detail in the hopes that it helps to improve the manuscript.\nWe thank the reviewer for taking the time to point out perceived issues with our data and analysis. There is indeed a\nwealth of data in this manuscript. We hope that with our revisions the significance of our results can now be properly\nappreciated.\nRNA and protein constructs:\n* It is often unclear what the RNA constructs used in each experiment exactly look like, these should be shown as\ninserts in each assay or minimally the complete set should be shown in Fig.3A.\nResponse:\nWe now show a complete set of RNA constructs used for RNA NMR in Fig 5 (previous Fig. 3A), and also added RNA\nconstructs (together with NMR CSP mapping when available) to all relevant Supplementary figures (6,8,9,14,16–\n22,24,25).\n* Why are deltaU constructs used? From the EMSA studies it seems that SL1-dIdeltaU binds much less well to\nHEXIM1 than SL1-dI (compare Suppl.Fig.5B gel1 and gel2). Yet, in ITC experiments only SL1-dI is used and not the\ncorresponding deltaU RNA. But then deltaU constructs are used for NMR. These are completely artificial constructs,\nwhy are they relevant?\nResponse:\nThe reviewer is correct that SLI-deltaU binds less well to Hexim1 than SL1-dI. We previously chose the deltaU\nconstruct to help with NMR assignments, since the wt-version (SL1-dI) causes even more severe line broadening for\nboth protein and RNA spectra. We have now included data from SL1-dI titrating with protein in Fig. 2 and\nSupplementary Fig. 6, and showed that the same Site1 is used for binding (Fig. 2C). We have also incorporated an\ninteresting comparison between SL1-dI-deltaU and SL1-dI CSP results (Fig. 2C and compare Supplementary Figs. 7\nand 11), which explains in a nucleotide-specific manner why a previous study observed a contribution of U63 bulge to\nHexim1 binding (Martinez-Zapien et al. NAR 2017), and also explains why Site1 is better than all the other sites we\nlooked at. Please see Results section “Characterization of Hexim monomer–7SK RNA Site1 interaction” (Lines 141–\n166) for details.\n*Why are different sets or RNAs used for EMSA vs. ITC vs. NMR? Why are different HEXIM1 constructs used for\nEMSA vs. ITC vs. NMR experiments? I understand that solubility is an issue for some protein constructs, which\nmakes it difficult to use them for NMR but for ITC and EMSA this should not be a problem, given that RNA affinities\nare in the nano to micro molar range. These differences make the manuscript not only confusing and difficult to\nfollow, they also draw comparisons and conclusions into question.\nResponse:\nWe used the same constructs for EMSA and NMR, in order to confirm binding stoichiometry. There are three longer\nRNA constructs used for NMR, as a part of the suite for divide-and-conquer strategy. The RNA constructs used are\nnow shown with the ITC data, which clearly shows that the same RNAs are used for NMR and ITC experiments\n(Supplementary Fig. 24). (We note that SL1-d(api), a construct studied previously by NMR (Pham et al. Commun Biol\n2022), is essentially the same as SL1-d, i.e. differs only in the apical loop.)\nRNA NMR experiments and CSPs:\n*p.8 states \"Using chemical shift perturbation (CSP) of RNA imino proton resonances in 1H-1H NOESY spectra in the\nabsence versus presence of monomeric Hexim1 BR-L-AR or BR, we identified SL1 Sites 1 and 2 as two high affinity\nbinding sites, and Sites 4 and 5 as two lower affinity binding sites (Fig. 3 and Supplementary Fig. 7).\"\n-> Several problems here: # How can CSPs inform on high/low affinity binding sites?\nResponse:\nFor the NMR studies, RNA binding sites were established using the shorter RNAs. Then for the longer RNAs that\ncontained more than one binding site, titration of the Hexim1 constructs shows clearly which site is occupied at 1:1\nratio (i.e. has higher affinity) based on the observed CSP. We have re-organized the relevant figure panels and\nadded small cartoons (now Fig. 5A) to better illustrate these direct comparisons. The binding site relative affinities\ndetermined by NMR are interpreted in conjunction with EMSA stoichiometric shifts (Supplementary Fig. 14).\nExchange behaviors on NMR titration timescale are also consistent with the relative order of affinities, where lower\naffinity binding shifts to faster exchange likely due to increased off-rate.\n# CSPs for RNA is never shown anywhere, only (difficult to distiguish) shades of blue in the base letters (Fig.3A,\nS5A) indicate them to some extent. CSPs should be shown in a graph analogous to protein CSPs.\nResponse:\nThe CSPs are shown on the secondary structures of the RNAs to make it easier for the reader to see where the\nbinding sites are. We now include individual Supplementary Figures for each RNA construct, their imino walk\nassignments, CSP plots, and 1D titration series (main Figs. 2 and 5, Supplementary Figs. 6, 8, 16–22).\n# NMR experiments are done with inconsistent protein constructs, which on top differ from what the sentence stated\ni.e. SL-dIdelaU with BR-L-AR, SL1-dII with BR-L, SL1-mdII with BRhalf. This means, BR is never used, instead BR-L\nand BRhalf were used but only for one RNA each. If both protein and RNA are changed in each experiment, how can\nconclusions about affinities to individual RNA binding sites be made?\nResponse:\nWe now show by direct comparison among BR-L-AR, BR-L, BR and BRARM1 (renamed from BRhalf for clarity)\nconstructs that the same ARM1 amino acids are perturbed by binding to Site1 RNA (main Fig. 2 and Supplementary\nFig. 10) for all these protein constructs. For longer constructs of RNA, BRARM1 is used to minimize line-broadening. A\ndirect comparison of RNA CSP between BR-L-AR and BRARM1 binding to Site1 nucleotides is shown in\nSupplementary Fig. 17D. In the longer RNA constructs with two or more sites, conclusions of relative order of binding\naffinity were drawn from each stand-alone titration experiment, not by comparisons across different RNA constructs\n(revised Fig. 5A). Quantitative comparison of binding affinities was done with ITC measurements, where the\nexperimental designs were informed by the NMR and EMSA experiments so that we know what binding event was\nbeing observed in each ITC titration.\n*p.8 states \"when using longer 7SK RNA constructs that include two adjacent binding sites, the higher affinity site of\nthe two is occupied by stoichiometric addition of Hexim1 BR as shown by NMR CSP of RNA iminos, i.e. in SL-d Site\n1 shows CSP instead of Site 4, in SL1-mdII Site 2 shows CSP instead of Site 4, and in SL1-pm Site 2 shows CSP\ninstead of Site 5 (Fig. 3A and Supplementary Fig. 5A);\"\n-> Where is this data? There is no SL1-d NMR dataset shown anywhere. Furthermore, binding site 4 in SL1-d in the\nscheme is shown in grey, which means \"not assigned\". So how can any conclusion about protein binding to this\nregion be made?\nResponse:\nWe now report non-exchangeable base and H1’ proton assignments for shorter RNA constructs, and provide\nevidence that the imino proton CSPs can capture the Hexim1 binding sites very well, when compared to CSPs from\nthe whole set of proton assignments (Fig. 2B, filled signs versus empty signs, and Supplementary Fig. 8C).\nRegarding SL1-d NMR data, as described 2 points above, we now include individual Supplementary Figures for each\nRNA construct, their imino walk assignments, CSP plots, and 1D titration series (SL1-d data shown in main Fig. 5,\nSupplementary Figs. 17). Regarding the gray residues, we have obtained additional assignments for imino and base\nprotons. We note that gray indicates not available CSP pairs, due to line broadening of bound spectrum or spectral\noverlap (now clarified in figure legends).\nEMSAs/ITC - Several issues with presentation, analysis and interpretation of EMSAs and ITC experiments:\n*Instead of reporting RNA-protein ratios, the EMSA binding assays should rather report concentrations. Kd values\nshould be calculated and compared to ITC data.\nResponse:\nThe concentrations used for EMSA were chosen to determine stoichiometry in conjunction with the NMR binding\nexperiments, not binding affinity, hence molar ratios were used. Therefore these EMSAs can only be analyzed for KD\napparent. For the reviewer, we fit the EMSA bands to KD,app values, also called microscopic dissociation constants, to\nassist in comparison across EMSA results. We have added “Notes for EMSA binding curve fitting” below\nSupplementary Fig. 14, which provide details for the calculations and Supplementary Fig. 15 for fitting results.\n*It is unclear how the assignments on the side of the EMSA gels with binding sites 1/2/4/5 can be made, especially\nfor SL1-ddeltaU, when both site 4 and site 1 were bound separately at roughly 0.9 RNA/protein ratio (compare\nSuppl.Fig.5B gel1 and gel3). All that can be concluded is that the first shift has probably 1 HEXIM1 bound and the\nsecond shift has probably 2 HEXIM1s bound to the RNA.\n*Similarly, statements in the text are not supported by the data. E.g. p.9 \"Indeed, EMSAs of SL1 constructs with full-\nlength Hexim1 show that Hexim1 dimer binds first to Sites 1 and 2 and subsequently to Sites 4 and 5 (Supplementary\nFig. 5D,E).\" All that can be concluded from this EMSA is that two proteins bind at 1.2 ratio and another two at 2.4\nratio. Which RNA sites they bind to cannot be seen in this assay.\nResponse:\nIt is true that sites cannot be determined from EMSA alone. However, as explained above, EMSA results were used\nin conjunction with NMR titration experiments to determine which site (high-affinity) is occupied at 1-to-1\nstoichiometry but not the other site (lower-affinity) out of two. We have now added NMR CSPs mapped on RNA\nconstructs next to each EMSA gel, and fit EMSA bands to KD,app to better illustrate these analyses. In addition, we\nhave obtained quantitative KD by ITC, as addressed below.\n*All ITC curves should be included in the supplement. Some of the listed affinities are comparably weak (micro molar\nKd) and fitting gives substoichiometry N-values, which could indicate that these curves do not reliably report N or Kd\nany more and should rather be given as approximate boundary values (rather than four significant digits).\nResponse:\nWe have now included all ITC curves in Supplementary Fig. 24. Direct ITC binding experiments can reliably\ndetermine KD within the range of 100µM to 1nM, i.e. our KD values fell well within this range (2.1µM to 73nM). For ITC\nexperiments, a 1-to-1 stoichiometric binding often corresponds to a N value smaller than 1.0 (“substoichiometry”), e.g.\nthe Calcium-EDTA control titration yields an N value of 0.7–0.8 on ITC. Since we did use the N values to compare\none-site to two-site binding, and to illustrate impaired release of Hexim1 autoinhibition, we also did the appropriate\ncontrol by doing the reverse ITC titrations by swapping protein from cell to syringe (less optimal due to more protein\naggregation at higher concentrations). These experiments gave N values that are approximately 1/N of the equivalent\ntitration pair in the original titration order. For example, SL1-dI was N=0.76, 1 over this N will be 1/0.76 = 1.32, and\nreverse order SL1-dI had N=1.12 (slightly smaller than 1.32, as anticipated from more protein aggregation). We have\nnow also cross-validated the accuracy of these N values by repeating key titrations with a newly acquired ITC\ninstrument from a different company (TA instrument, Affinity ITC), and acquired the same N numbers as the previous\ninstrument we used (Malvern, MicroCal ITC).\nRegarding the reported values and errors, they are average and standard deviations from three or more independent\nITC titration experiments, not just fitting error from one experiment (which is sometimes reported as boundary\nvalues). We have included this clarification in Fig. 7 and Supplementary Fig. 24 captions. The decimal places are\nkept identical, in order to assist fast comparison between weak and strong binding sites, but we have rounded up the\nweaker binders to three significant figures to reflect the three significant figures of ITC instrument reported values.\n.\nPRE experiments and PYNT autoinhibition model:\n* In Fig. 4B-E regions with PRE intensity ratios below mean-0.5sigma and mean-1sigma are supposedly shaded in\norange and red. Yet in almost every one of the shaded regions several residues lie above the supposed cut-off (or\nare simply not assigned) and conversely in the white regions several residues have lower than cut-off ratios (or are\nsimply not assigned). Thus, the shaded areas give an oversimplified and scewed picture of the data and this is\ncemented further by the structure cartoons presented on top. Based on the data one could equally well argue that the\nAR is in transient contact with any one of the other regions of HEXIM1. Additional and complementary evidence\nwould be required to make this section convincing.\nResponse:\nWe have clarified in the text how the regions of mean-0.5sigma and mean-1sigma PREs were defined, i.e. these\ncorrespond to stretches with three or more non-contiguous residues, in order to assist with identifying which structural\nelements are involved. We have revised and enlarged the cartoons to better illustrate the PRE interpretation.\nFor complementary evidence of autoinhibition, we have added ITC data on AR and PYNT mutants (A3m, A12m and\nDeltaPYNT) in full-length Hexim1 dimer, to further support our model of inter-monomer autoinhibition. These mutants\nall turned full-length Hexim1 into a non-specific RNA binder, illustrating their significant roles in autoinhibition and\nRNA binding (Lines 364–370, Fig. 7 and Supplementary Fig. 25). We also obtained NativePAGE and mass\nphotometry data that provide further support for the AR–BR centric inter-monomer interactions (Lines 109–126, Fig. 1\nand Supplementary Fig. 4).\n* On top of the weak data, for these experiments it is especially relevant that they are carried out in the context of a\ntruncated HEXIM1 that cannot form the obligate dimer. The N-terminal HEXIM1 regions could equally well bind back\nto the coiled-coil section, which would also lead to reduced RNA affinity of full length HEXIM1 for 7SK RNA (as\ndetermined by ITC).\nResponse:\nWe cannot completely rule out a potential weak interaction between Hexim1 N-term and coiled-coil domain (also see\ndiscussion of autoinhibitory N-terminus below), but we have acquired new mass photometry data on mixed\ncomplexes between L-AR-CC and BR-L-AR, and between Hexim1 and BR-L-AR, which are consistent with BR-L-AR\ncentric inter-monomer interactions. The fact that L-AR-CC construct does not form higher order complex at 1:4 molar\nratio rules out any stable interaction between the coiled-coil domain and BR-L-AR at 40nM concentration.\n* If the PYNT motif is truely involved in reducing RNA affinity and HEXIM1 making binding \"more selective\", then a\ndeletion of this short motif should increase RNA affinity of HEXIM1 and make it \"less selective\".\nPlease see our response (2 points above) where we describe new AR and PYNT mutants (A3m, A12m and\nDeltaPYNT) that turn Hexim1 into non-specific binder.\n* Conceptually the model also poses a problem as most of HEXIM1 in cells is bound to dsRNA (Li et al. NAR 2007),\nand should therefore be relieved of the autoinhibited conformation and associate with P-TEFb according to the\nproposed model, but this seems not to be the case in cells. Furthermore, in previous studies the very N-terminus that\nis not studied here (aa 1-120) has an auto-inhibitory effect (Li et al. JBC 2005).\nResponse:\nOur NBLA construct spans residues 1–253, including the very N-terminus. Therefore, any difference observed\nbetween NBLA monomer and Hexim1 dimer by ITC, is only due to the addition of coiled-coil dimerization domain, not\ndue to a lack of N-terminus. That said, our model does not exclude the auto-inhibitory effect of the N-terminus aa 1-\n120, but we cannot identify the responsible N-terminal elements, if any, by looking at the sequence alignment of\nHexim1 and Hexim2 orthologs. For human Hexim1 sequence, its extreme N-terminus contains a slightly acidic and\nhydrophobic stretch (residues 1-26, net -4 charge, or -8 charge for residues 1-66), but the acidic residues are not\nclustered, rather this segment appears more amphipathic than acidic. In comparison, central region AR (-13 total net\ncharges) contains clustered net -6 charges for n-AR, and clustered net -10 charges for c-AR. It was also interesting\nthat in Li et al. JBC 2005, the DeltaN construct could still respond to 7SK RNA binding, which significantly enhanced\nits P-TEFb-inhibitory activity, consistent with releasing BR-L-AR mediated autoinhibition. We note in our revised\nmanuscript that the inclusion of N-terminus increased the solubility of RNP during ITC titrations, pointing to a\ncontribution of the disordered N-terminus to an altered protein biochemical property. It is possible that the extreme N-\nterminus could provide further stabilization to the BR–PYNT–AR interaction of the central region or contact CC\ndomain. Due to length constraint, we added these discussions to “Supplementary discussion regarding N-terminus of\nHexim and Tat” below Supplementary Fig. 26.\nPlease see our response (3 points above) where we describe new AR and PYNT mutants (A3m, A12m and\nDeltaPYNT) that turn Hexim1 into non-specific binder.\nRegarding Hexim bound to dsRNA in cells, from our comparison of the five mapped RNA sites, we conclude that the\ndetermining factors for high-affinity Hexim binding site are an A-U rich stem with at least one flanking U-rich bulge at\nthe 5’ side, which will inform future studies of Hexim interactions with other cellular targets. Please see the refined\nmodel figure (Fig. 8) and Discussion third paragraph (Lines 402–414) for illustration and description of the issue of\ngeneral Hexim1–RNA specificity. We would like to also mention a recently study published during our revision\nprocess (Graham et al. Mol Cell 2025) for live cell fluorescent imaging of 7SK RNP proteins, which showed that the\nportion of Hexim1 not bound to 7SK RNP exhibited a fast-diffusing peak (6-7 μm2/s versus 2-3 μm2/s peak for CycT1-\nand Larp7-bound peak), consistent with our “facilitated search” model, which implies that the pool of 7SK-free Hexim1\nlikely does not stably associate with other cellular dsRNA targets (unless these dsRNA targets contain a similar high-\naffinity site to what we determined for 7SK). We note that Li et al. NAR 2007 showed a successful enrichment of 7SK,\nnot U2 and U6 RNA, through affinity pull down of Hexim1, illustrating specificity. It is possible that mir-16-1 microRNA\nenriched by the Hexim1 pull-down in Li et al. NAR 2007 is small and thus compatible with the 6-7 μm2/s fast-diffusing\npeak observed in Graham et al. Mol Cell 2025. These interesting observations further highlight the significance of our\ncurrent study where we dissected Hexim–RNA binding specificity.\nFigure citations - The text jumps back and forth between figures some in the wrong order and some figure citations\nseem to be entirely wrong e.g. Fig. 2B should be cited on p.5 (\"...referred to as BR-L-AR in the following text.\") before\nFig. 2A is coming in. There is no Fig.3E but it is referred to in the text (p.7 \"...bulge (U40U41) is also present 3’ to the\nalternative GAUC palindrome (Fig. 3E).\")\nResponse:\nWe have fixed these errors in the revised manuscript.\nChemical exchange - If this paper is meant for a general audience, the authors should explain what slow,\nintermediate and fast exchange mean in context of the system studied and the complexes that are formed here (e.g.\non p.6, p.8).\nResponse:\nDue to the manuscript length constraint, we have modified relevant text to give a brief explanation and cited a general\nreview article for this purpose.\nAbstract final sentence \"These results provide mechanistic insights into Hexim–RNA specificity and explain how P-\nTEFb can be effectively regulated to respond to changing levels of transcriptional signaling.\" - This conclusion is not\nwarranted given that the authors have not carried out any assays in cells and used only artificial in vitro systems with\nfew (and mostly severely truncated) components.\nResponse:\nWe have now separated the conclusion sentence and the broader implication sentence into two stand-alone\nsentences to avoid this confusion.\nFigure inconsistencies - I understand that some peaks are broadened upon addition of RNA but it still looks like some\nbars are missing in Suppl.Fig.4C basic region in comparison to Fig. 2D/E? Also the baseline at 0 is missing in\nSuppl.Fig.4C.\nResponse:\nSupplementary Fig 4C is secondary structural propensity scores, calculated from C-alpha and C-beta chemical shifts\n(see Methods for detail). The two residues that we could assign amide chemical shifts in Fig. 2C, however, could not\nbe assigned both C-alpha and C-beta chemical shifts for the SSP calculation. We used carbonyl assignments to\nverify the amide resonances. Clarification of these and a line of zero SSP value have been added to Supplementary\nFig. 5C and caption.\nSuppl. Fig. 1 and 2: The sequence alignments seem to be cut out of larger alignments because they display\nnumerous empty columns that hold no information. These empty columns should be removed to make the alignments\nmore compact and easier to understand.\nResponse:\nYes indeed, we performed sequence alignments with 294 genes for Hexim1 and 210 genes for Hexim2 from NCBI\ngene cards, and randomly selected three genes each, in addition to human and mouse, for display (5 out of 294 for\nHexim1 and 5 out of 210 for Hexim2). This information was described in Methods section titled “Multiple sequence\nalignment” for the details of alignment analysis. Many of the empty columns reflect differences between Hexim1 and\nHexim2 paralogues. Other empty columns are important for us to understand which regions have insertions from\northologs, which also inform on whether adjacent structural elements are more separate modules or belong to a\ncontinuous domain. We have manually removed the empty columns resulting from full alignment insertions from the\nfive representative gene displays, which mainly shrunk Supplementary Fig. 1, not much for Supplementary Fig. 2.\nWe have added these now missing details of the insertions to Supplementary Fig. 1 caption."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The manuscript has been significantly improved, although it remains rather complicated to read in places. However, this is probably unavoidable when it comes to the description of NMR chemical shift perturbation data and the explanation of MTSL spin labelling experiments. The use of RNA constructs is a bit better to follow now and the confusion with mixed constructs in neighbouring panels is partly resolved. The discussion benefits a lot from the new model figure (Fig. 8) of the proposed regulation of P-TEFb by the 7SK snRNP in the main text. The figures are well prepared, although they may be difficult to follow for non-NMR specialists.\nI have no further comments to this expert study.", "reviewer_3": "A few points to consider revising:\nReview:\n- Constructs were clarified reasonable, paper still hard to read\n- Fig. 1E: Where do percentages come from? How are the actual Mass-Photometry size correlated to the thought to be observed species… not clear\n- According to native gel (Fig. S4a), Hexim1 can also form tetramers. not indicated, please clarify\n- The other species in the mass photometry studies are ignored? Why, please explain\no Fig. S4c/1E the calculation of trimer and tetramer are not evident\no Which of the data for BR-L-AR is for 40 nM or 4-25µM?\n- CSP mean doesn’t make sense – one usually establishes CSP baseline that is not changing, and anything bigger than the std of this is considered a change in CSP… so with mean you are already excluding some relevant CSPs."}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript has been significantly improved, although it remains rather complicated to read\nin places. However, this is probably unavoidable when it comes to the description of NMR\nchemical shift perturbation data and the explanation of MTSL spin labelling experiments. The\nuse of RNA constructs is a bit better to follow now and the confusion with mixed constructs in\nneighbouring panels is partly resolved. The discussion benefits a lot from the new model figure\n(Fig. 8) of the proposed regulation of P-TEFb by the 7SK snRNP in the main text. The figures\nare well prepared, although they may be difficult to follow for non-NMR specialists.\nI have no further comments to this expert study.\nResponse: We thank the reviewer for the positive feedback and for acknowledging our efforts\nto improve readability for non-NMR specialists. During the final revision, we have further refined\nthe NMR sections to improve flow and readability, while preserving technical precision:\n(1) We have modified Fig. 3 and Fig. 4 to specifically indicate the Pro residues and overlapping\nresidues, and Fig. 6C by adding domain diagram.\n(2) We revised the NMR-dense paragraphs lightly to improve the flow while maintaining the\nsame technical details (Lines 142-161 and Lines 187-219).\nReviewer #3 (Remarks to the Author):\nA few points to consider revising:\nReview:\n- Constructs were clarified reasonable, paper still hard to read\nResponse: We appreciate the reviewer’s feedback. Please see response for Reviewer #1\nregarding further edits we made make the paper easier to read.\n- Fig. 1E: Where do percentages come from? How are the actual Mass-Photometry size\ncorrelated to the thought to be observed species… not clear\nResponse: The percentages are derived from the Mass Photometry analysis, which calculates\nthe proportion of total particle counts represented by the Gaussian-fitted peaks. For instance,\n~71% of total particle counts fall under the fitted peak corresponding to 49 ± 10.2 kDa for the\nBR-L-AR construct, while ~29% correspond to larger particles in the 60–160 kDa range. These\nobservations are consistent with the presence of higher-order oligomers detected by native\nPAGE. This detail about percentages has been incorporated into Methods and Fig. S4 caption.\nTo further clarify this, we have revised the figure caption for Fig. 1E as follows:\n“The observed Mass Photometry peak corresponds to a molecular weight of 49 ± 10.2 kDa—\nbetween the theoretical trimer (39.6 kDa) and tetramer (52.8 kDa) sizes, but closer to the latter.\nThis shift likely results from the trimer peak being near the 30 kDa detection limit (gray arrow),\ncausing partial peak truncation and overlap with the tetramer peak. Monomer and dimer species\nfall below the detection threshold and are not observed.”\n- According to native gel (Fig. S4a), Hexim1 can also form tetramers. not indicated, please\nclarify\nResponse: We have now mentioned the minor tetrameric species of full-length Hexim1\nobserved by native PAGE at 4 µM protein concentration in the Fig. S4A caption (6.7% tetramer\nspecies by counting gel band intensities). This contrasts with the L-AR-CC construct, which\nlacks the BR and therefore does not form tetramers at 8 µM. At 40nM dilute condition, no\ntetramer species of Hexim1 were detected by Mass Photometry (Fig. S4E). The dimer bands\nbeing the major bands in native PAGE is now clarified in the main text (line 117).\n- The other species in the mass photometry studies are ignored? Why, please explain\no Fig. S4c/1E the calculation of trimer and tetramer are not evident\no Which of the data for BR-L-AR is for 40 nM or 4-25µM?\nResponse: All Mass Photometry data were collected at 40 nM protein concentration. The\nconcentrations and the higher-order oligomeric species (could not be fit well to gaussian\nfunctions due to their lower counts at 40nM and apparent heterogeneity) are now labeled on the\nMass Photometry panels (Fig. 1E and Fig. S4). Details regarding BR-L-AR trimer and tetramer\nspecies are provided two points above.\n- CSP mean doesn’t make sense – one usually establishes CSP baseline that is not changing,\nand anything bigger than the std of this is considered a change in CSP… so with mean you are\nalready excluding some relevant CSPs.\nResponse: One or two standard deviations above the mean CSP value across the entire\nprotein is a widely accepted threshold for identifying residues with significant perturbations. This\napproach is appropriate here, given that several BR residues broaden beyond detection upon\nRNA binding—likely leading to underestimation of the largest CSP values.\nWe agree that residues showing weaker or transient interactions may fall below the mean+STD\ncutoff. To capture these subtler effects, we plotted both mean and mean+STD thresholds for\nprotein CSPs (Fig. 2E) and three-tier thresholds (mean, mean+0.5×STD, and mean+STD) for\nRNA CSPs (Fig. 1B, Fig. 5). For instance, the PYNT motif (residues 202–209) displays CSPs\nbetween mean and mean+STD, consistent with its proposed transient intermolecular\ninteractions with fast exchange dynamics in our study. That said, we chose to stay conservative\nwith the main text description, due to the likely underestimation of the largest CSP."}]

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