Characterizing RNA dynamics at atomic resolution using solution-state NMR spectroscopy

Bothe, Jameson R.; Nikolova, Evgenia N.; Eichhorn, Catherine D.; Chugh, Jeetender; Hansen, Alexandar L.; Al‐Hashimi, Hashim M.

doi:10.1038/nmeth.1735

Cited by 148 publications

(151 citation statements)

References 98 publications

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“…Whereas R 1 and R 1 ρ report on ps-ns dynamics, RDCs have an extended timescale resolution and are sensitive to internal motions ranging from ps to ms, providing insight into dynamics occurring on an ns-μs timescale (48)(49)(50)(51)(52)(53)(54). We performed order tensor analysis (55, 56) using the RAMAH program (57) to compute the generalized degree of order (GDO; ϑ) (58) for each helix.…”

Section: Resultsmentioning

confidence: 99%

Structural determinants for ligand capture by a class II preQ ₁ riboswitch

Kang

Eichhorn²,

Feigon

2014

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

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Prequeuosine (preQ 1 ) riboswitches are RNA regulatory elements located in the 5′ UTR of genes involved in the biosynthesis and transport of preQ 1 , a precursor of the modified base queuosine universally found in four tRNAs. The preQ 1 class II (preQ 1 -II) riboswitch regulates preQ 1 biosynthesis at the translational level. We present the solution NMR structure and conformational dynamics of the 59 nucleotide Streptococcus pneumoniae preQ 1 -II riboswitch bound to preQ 1 . Unlike in the preQ 1 class I (preQ 1 -I) riboswitch, divalent cations are required for high-affinity binding. The solution structure is an unusual H-type pseudoknot featuring a P4 hairpin embedded in loop 3, which forms a three-way junction with the other two stems. 13 C relaxation and residual dipolar coupling experiments revealed interhelical flexibility of P4. We found that the P4 helix and flanking adenine residues play crucial and unexpected roles in controlling pseudoknot formation and, in turn, sequestering the Shine-Dalgarno sequence. Aided by divalent cations, P4 is poised to act as a "screw cap" on preQ 1 recognition to block ligand exit and stabilize the binding pocket.Comparison of preQ 1 -I and preQ 1 -II riboswitch structures reveals that whereas both form H-type pseudoknots and recognize preQ 1 using one A, C, or U nucleotide from each of three loops, these nucleotides interact with preQ 1 differently, with preQ 1 inserting into different grooves. Our studies show that the preQ 1 -II riboswitch uses an unusual mechanism to harness exquisite control over queuosine metabolism.R iboswitches are functional noncoding RNA elements often found at the 5′ end of genes that bind to effector molecules, usually metabolites, to attenuate gene expression (1-8). They generally contain two modular elements, an aptamer that binds directly to a cognate ligand, followed by an expression platform that carries out the regulatory function. Ligand binding induces a conformational rearrangement in the aptamer domain, which is transduced to the expression platform, turning gene expression on or off at the transcription or translation level.Structural studies of diverse riboswitches bound to their cognate ligands have revealed that they have evolved various strategies for high-affinity and specific binding between RNA receptors and ligands. In general, the aptamer domain enfolds the ligand, forming a highly complex 3D conformation using a host of tertiary interactions, including base triples, kink turns, ribose zippers, A-minor motifs, loop-loop interactions, and pseudoknots (9-11). Some riboswitches require divalent cations for ligand binding (9, 12-16), whereas in others divalent cations stabilize the bound state (17-21). Some ligands, including S-adenosyl methionine (22), S-adenosyl homocysteine (22, 23), cyclic di-GMP (24, 25), and prequeuosine (preQ 1 ) (26, 27), have more than one class of riboswitch, with each class adopting a different 3D fold to achieve recognition of the same ligand.Two classes of riboswitches that bind preQ 1 , a precursor t...

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Section: Resultsmentioning

confidence: 99%

Structural determinants for ligand capture by a class II preQ ₁ riboswitch

Kang

Eichhorn²,

Feigon

2014

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

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“…In the E-SS construct, the adjoined helix helps decouple internal and overall motions and provides a reference for assessing overall motions, making it easier to measure the absolute level of motions in the ssRNA tail. This domain-elongation strategy has widely been applied in studies of RNA systems, but never for an ssRNA tail (Getz et al 2007a;Bothe et al 2011).…”

Section: Spin Relaxationmentioning

confidence: 99%

Structural dynamics of a single-stranded RNA–helix junction using NMR

Eichhorn

Al‐Hashimi

2014

RNA

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Many regulatory RNAs contain long single strands (ssRNA) that adjoin secondary structural elements. Here, we use NMR spectroscopy to study the dynamic properties of a 12-nucleotide (nt) ssRNA tail derived from the prequeuosine riboswitch linked to the 3 ′ end of a 48-nt hairpin. Analysis of chemical shifts, NOE connectivity, 13 C spin relaxation, and residual dipolar coupling data suggests that the first two residues (A25 and U26) in the ssRNA tail stack onto the adjacent helix and assume an ordered conformation. The following U26-A27 step marks the beginning of an A 6 -tract and forms an acute pivot point for substantial motions within the tail, which increase toward the terminal end. Despite substantial internal motions, the ssRNA tail adopts, on average, an A-form helical conformation that is coaxial with the helix. Our results reveal a surprising degree of structural and dynamic complexity at the ssRNA-helix junction, which involves a fine balance between order and disorder that may facilitate efficient pseudoknot formation on ligand recognition.

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“…Furthermore, NMR has been widely applied in clinical research, called magnetic resonance imaging (MRI), to help physicians diagnose potential disease regions in soft tissues, such as human brains (91). Furthermore, other than proteins, NMR may be also applied to study the dynamics of ribonucleic acid (RNA) (92), as well as the structure and conformational changes of carbohydrates (93).…”

mentioning

confidence: 99%

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

Sun¹,

Kale²,

Azurmendi³

et al. 2013

MPMI

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Oomycetes, such as Phytophthora sojae, are plant pathogens that employ protein effectors that enter host cells to facilitate infection. Plants may overcome infection by recognizing pathogen effectors via intracellular receptors (R proteins) that form part of their defense system. Entry of some effector proteins into plant cells is mediated by conserved RxLR motifs in the effectors and phosphoinositides (PIPs) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effector proteins involved in PIP recognition. To clarify these differences, I have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5).

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Characterizing RNA dynamics at atomic resolution using solution-state NMR spectroscopy

Cited by 148 publications

References 98 publications

Structural determinants for ligand capture by a class II preQ ₁ riboswitch

Structural determinants for ligand capture by a class II preQ ₁ riboswitch

Structural dynamics of a single-stranded RNA–helix junction using NMR

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

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Characterizing RNA dynamics at atomic resolution using solution-state NMR spectroscopy

Cited by 148 publications

References 98 publications

Structural determinants for ligand capture by a class II preQ 1 riboswitch

Structural determinants for ligand capture by a class II preQ 1 riboswitch

Structural dynamics of a single-stranded RNA–helix junction using NMR

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

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Structural determinants for ligand capture by a class II preQ ₁ riboswitch

Structural determinants for ligand capture by a class II preQ ₁ riboswitch