Argininamide Binding Arrests Global Motions in HIV-1 TAR RNA: Comparison with Mg2+-induced Conformational Stabilization

Pitt, Stephen W.; Majumdar, Ananya; Serganov, Alexander; Patel, Dinshaw J.; Al‐Hashimi, Hashim M.

doi:10.1016/j.jmb.2004.02.031

Cited by 79 publications

(146 citation statements)

References 51 publications

(91 reference statements)

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“…In contrast, the equivalent A22-U40 base pair in TAR forms only after ligand binding. 23 No significant bend was observed between the two stems which are arranged in near-coaxial stacking as is evident by standard stacking NOEs between C304 to G305. Tertiary contacts in the bulge, for example a cross-strand NOE between a G305 N2 amino proton and the G322 H8 proton as well as our dipolar coupling data define a 60° inter-helical displacement between the two adjacent stems.…”

Section: Structure Of 7sk-sl4 Identifies a Preformed Protein-binding mentioning

confidence: 84%

“…17,18 Several NMR studies have elucidated the major conformational transition that argininamide (a tight-binding analog of arginine) induces in the TAR structure upon complex formation. [19][20][21][22][23] In the free form the bulged residues and the terminal A22 and U40 residues are not involved in hydrogen bonding interactions but upon arginine binding U23 and A22 sandwich arginine by forming a critical U23:A27-U38 base-triple and a A22-U40 base pair (Figure 1a, dashed red lines) and leads to a global dampening of interhelical motions. 22,23 The conserved stem loop 4 of 7SK (7SK-SL4) presents significant sequence homology to the TAR RNA from HIV-1 ( Figure 1a).…”

Section: Introductionmentioning

confidence: 99%

“…[19][20][21][22][23] In the free form the bulged residues and the terminal A22 and U40 residues are not involved in hydrogen bonding interactions but upon arginine binding U23 and A22 sandwich arginine by forming a critical U23:A27-U38 base-triple and a A22-U40 base pair (Figure 1a, dashed red lines) and leads to a global dampening of interhelical motions. 22,23 The conserved stem loop 4 of 7SK (7SK-SL4) presents significant sequence homology to the TAR RNA from HIV-1 ( Figure 1a). As an initial step towards understanding the structural basis of 7SK function and to draw parallels with retroviral TAR, we determined the NMR solution structure of 7SK-SL4 both free and in complex with argininamide.…”

Section: Introductionmentioning

confidence: 99%

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Preformed Protein-binding Motifs in 7SK snRNA: Structural and Thermodynamic Comparisons with Retroviral TAR

Durney

D'Souza

2010

Journal of Molecular Biology

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Section: Structure Of 7sk-sl4 Identifies a Preformed Protein-binding mentioning

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Preformed Protein-binding Motifs in 7SK snRNA: Structural and Thermodynamic Comparisons with Retroviral TAR

Durney

D'Souza

2010

Journal of Molecular Biology

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“…To test this possibility, we examined whether binding of argininamide (ARG), a ligand mimic of TAR's cognate protein target Tat, to the TAR bulge affects relaxation dispersion measured in the apical loop. Previous NMR studies (25,44) showed that ARG binds TAR in and around the bulge stabilizing a coaxial conformation in which bulge residue U23 forms a reverse Hoogsteen base pair with A27-U38 in the upper stem. If our proposed ES2 structure is correct, one would expect that locking the bulge conformation upon ARG binding would quench all relaxation dispersion arising due to exchange with ES2, including in the apical loop.…”

Section: G34-c8mentioning

confidence: 98%

Invisible RNA state dynamically couples distant motifs

Lee

Dethoff

Al‐Hashimi

2014

Proc. Natl. Acad. Sci. U.S.A.

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113

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Using on-and off-resonance carbon and nitrogen R1ρ NMR relaxation dispersion in concert with mutagenesis and NMR chemical shift fingerprinting, we show that the transactivation response element RNA from the HIV-1 exists in dynamic equilibrium with a transient state that has a lifetime of ∼2 ms and population of ∼0.4%, which simultaneously remodels the structure of a bulge, stem, and apical loop. This is accomplished by a global change in strand register, in which bulge residues pair up with residues in the upper stem, causing a reshuffling of base pairs that propagates to the tip of apical loop, resulting in the creation of three noncanonical base pairs. Our results show that transient states can remodel distant RNA motifs and possibly give rise to mechanisms for rapid long-range communication in RNA that can be harnessed in processes such as cooperative folding and ribonucleoprotein assembly.NMR spectroscopy | dynamics | R1ρ relaxation dispersion | nucleic acids I t is now well-established that RNA sequences do not code for a single static structure, but rather, many conformations that populate energetic minima along a free-energy landscape (1, 2). Cellular inputs, ranging from changes in temperature and pH to the binding of proteins, other RNAs, and ligands, can preferentially stabilize select conformations along the landscape, resulting in dynamic changes in RNA structure that drive the multistep catalytic cycles of ribozymes (3), regulatory activities of riboswitches (4) and other RNA-based switches (5), and the dynamic assembly and disassembly of ribonucleoprotein (RNP) complexes (6).A common mode of RNA dynamics involves rearrangements in secondary structure that can melt or create entire hairpins, and thereby expose or sequester key regulatory elements that are several nucleotides long (1,4,7,8). Such secondary structural transitions entail large kinetic barriers, so they are often catalyzed by RNA-binding proteins (9), ATP-dependent chaperones (10), or otherwise occur by modulating cotranscriptional folding (5, 11). Recently, NMR R1ρ relaxation dispersion experiments (12-15) in concert with mutagenesis (16) have helped uncover more labile RNA secondary structural transitions that can take place without assistance from external cofactors at rates that are 2-4 orders of magnitude faster than larger-scale secondary structural rearrangements. These transitions entail excursions away from the energetically favorable ground state (GS) toward lowpopulated (typically populations <15%) and short-lived (lifetime < milliseconds) species often referred to as "excited states" (ES) (12, 13). These invisible RNA ES feature localized reshuffling of base pairing in and around noncanonical motifs such as bulges, internal loops, and apical loops (16) which can also expose or sequester functionally important residues or promote ATPindependent large-scale changes in secondary structure (14, 15). These faster and more localized changes in secondary structure may meet unique demands in RNA-based regulatory functions (16).Using...

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“…These characteristics are the A22:U40 (17, 37) and C30:G34 (17, 38, 39) base pairs; the U23:A27:U38 base triplet (16,17,19,40,41); and the extent of base stacking between the bulge residues U23, C24, and U25 (16,17,37). The CVs for the base pairs and triplet were implemented in a way that measures the number of hydrogen bonds between the residues involved in the interaction.…”

Section: Methodsmentioning

confidence: 99%

Structure of a low-population binding intermediate in protein-RNA recognition

Borkar

Bardaro

Camilloni

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The interaction of the HIV-1 protein transactivator of transcription (Tat) and its cognate transactivation response element (TAR) RNA transactivates viral transcription and represents a paradigm for the widespread occurrence of conformational rearrangements in protein-RNA recognition. Although the structures of free and bound forms of TAR are well characterized, the conformations of the intermediates in the binding process are still unknown. By determining the free energy landscape of the complex using NMR residual dipolar couplings in replica-averaged metadynamics simulations, we observe two low-population intermediates. We then rationally design two mutants, one in the protein and another in the RNA, that weaken specific nonnative interactions that stabilize one of the intermediates. By using surface plasmon resonance, we show that these mutations lower the release rate of Tat, as predicted. These results identify the structure of an intermediate for RNA-protein binding and illustrate a general strategy to achieve this goal with high resolution.RNA structure | NMR spectroscopy | metadynamics | exact RDC restraints | tensor-free method E ssentially all biochemical reactions taking place in living organisms are associated with macromolecular recognition events. A full understanding the molecular mechanisms underlying such events requires the characterization of binding intermediates, which are states that typically have lifetimes of less than a millisecond and may comprise only 5-15% of the conformational space of proteins (1) and nucleic acids (2, 3). Protein-protein and protein-DNA intermediates have recently been characterized at high resolution (4, 5), but despite considerable advances (3, 6-8), high-resolution structures for protein-RNA intermediates have not been reported yet.To address this problem, we focused on the well-studied process by which HIV, like other lentiviruses, hijacks the host transcription machinery to activate transcription of the viral genome (9-13). In HIV, transactivation (Fig. S1) requires binding of the transactivator of transcription (Tat) protein and the host positive transcription elongation factor b (P-TEFb) complex (11) to the transactivation response element (TAR), a 59-residue RNA stem-loop ( Fig. 1 and Fig. S1) with a highly dynamic structure (10, 12, 13). The NMR structures of free TAR (14-16) and of TAR bound to peptide fragments of Tat and to peptide mimetics of and other lentiviruses (21,22) revealed the conformational properties of TAR in its free and bound states, and demonstrated that this RNA molecule undergoes significant dynamic rearrangements associated with its functions. Although the TAR-Tat complex has become a paradigm for the widespread occurrence of conformational rearrangements and molecular adaptation in protein-RNA recognition, the pathway and intermediates linking the free and bound states of TAR are still unknown. Results and DiscussionDetermination of the Tat-TAR Free Energy Landscape. Following an approach recently described for proteins (4) we ident...

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Argininamide Binding Arrests Global Motions in HIV-1 TAR RNA: Comparison with Mg2+-induced Conformational Stabilization

Cited by 79 publications

References 51 publications

Preformed Protein-binding Motifs in 7SK snRNA: Structural and Thermodynamic Comparisons with Retroviral TAR

Preformed Protein-binding Motifs in 7SK snRNA: Structural and Thermodynamic Comparisons with Retroviral TAR

Invisible RNA state dynamically couples distant motifs

Structure of a low-population binding intermediate in protein-RNA recognition

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