Search

La-related protein 7 (LARP7) are a family of RNA chaperones that protect the 3'-end of RNA and are components of specific ribonucleoprotein complexes (RNP). In Tetrahymena thermophila telomerase, LARP7 protein p65 together with telomerase reverse transcriptase (TERT) and telomerase RNA (TER) form the core RNP. p65 has four known domains-N-terminal domain (NTD), La motif (LaM), RNA recognition motif 1 (RRM1), and C-terminal xRRM2. To date, only the xRRM2 and LaM and their interactions with TER have been structurally characterized. Conformational dynamics leading to low resolution in cryo-EM density maps have limited our understanding of how full-length p65 specifically recognizes and remodels TER for telomerase assembly. Here, we combined focused classification of Tetrahymena telomerase cryo-EM maps with NMR spectroscopy to determine the structure of p65-TER. Three previously unknown helices are identified, one in the otherwise intrinsically disordered NTD that binds the La module, one that extends RRM1, and another preceding xRRM2, that stabilize p65-TER interactions. The extended La module (αN, LaM and RRM1) interacts with the four 3' terminal U nucleotides, while LaM and αN additionally interact with TER pseudoknot, and LaM with stem 1 and 5' end. Our results reveal the extensive p65-TER interactions that promote TER 3'-end protection, TER folding, and core RNP assembly and stabilization. The structure of full-length p65 with TER also sheds light on the biological roles of genuine La and LARP7 proteins as RNA chaperones and core RNP components., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Shq1 is an essential protein involved in the early steps of biogenesis and assembly of H/ACA ribonucleoprotein particles (RNPs). Shq1 binds to dyskerin (Cbf5 in yeast) at an early step of H/ACA RNP assembly and is subsequently displaced by the H/ACA RNA. Shq1 contains an N-terminal CS and a C-terminal Shq1-specific domain (SSD). Dyskerin harbors many mutations associated with dyskeratosis congenita. Structures of yeast Shq1 SSD bound to Cbf5 revealed that only a subset of these mutations is in the SSD binding site, implicating another subset in the putative CS binding site. Here, we present the crystal structure of human Shq1 CS (hCS) and the nuclear magnetic resonance (NMR) and crystal structures of hCS containing a serine substitution for proline 22 that is associated with some prostate cancers. The structure of hCS is similar to yeast Shq1 CS domain (yCS) and consists of two β-sheets that form an immunoglobulin-like β-sandwich fold. The N-terminal affinity tag sequence AHHHHHH associates with a neighboring protein in the crystal lattice to form an extra β-strand. Deletion of this tag was required to get spectra suitable for NMR structure determination, while the tag was required for crystallization. NMR chemical shift perturbation (CSP) experiments with peptides derived from putative CS binding sites on dyskerin and Cbf5 revealed a conserved surface on CS important for Cbf5/dyskerin binding. A HADDOCK (high-ambiguity-driven protein-protein docking) model of a Shq1-Cbf5 complex that defines the position of CS domain in the pre-H/ACA RNP was calculated using the CSP data., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The Saccharomyces cerevisiae RNase III enzyme Rnt1p preferentially binds to double-stranded RNA hairpin substrates with a conserved (A/u)GNN tetraloop fold, via shape-specific interactions by its double-stranded RNA-binding domain (dsRBD) helix α1 to the tetraloop minor groove. To investigate whether conformational flexibility in the dsRBD regulates the binding specificity, we determined the backbone dynamics of the Rnt1p dsRBD in the free and AGAA hairpin-bound states using NMR spin-relaxation experiments. The intrinsic microsecond-to-millisecond timescale dynamics of the dsRBD suggests that helix α1 undergoes conformational sampling in the free state, with large dynamics at some residues in the α1-β1 loop (α1-β1 hinge). To correlate free dsRBD dynamics with structural changes upon binding, we determined the solution structure of the free dsRBD used in the previously determined RNA-bound structures. The Rnt1p dsRBD has an extended hydrophobic core comprising helix α1, the α1-β1 loop, and helix α3. Analysis of the backbone dynamics and structures of the free and bound dsRBD reveals that slow-timescale dynamics in the α1-β1 hinge are associated with concerted structural changes in the extended hydrophobic core that govern binding of helix α1 to AGAA tetraloops. The dynamic behavior of the dsRBD bound to a longer AGAA hairpin reveals that dynamics within the hydrophobic core differentiate between specific and nonspecific sites. Mutations of residues in the α1-β1 hinge result in changes to the dsRBD stability and RNA-binding affinity and cause defects in small nucleolar RNA processing invivo. These results reveal that dynamics in the extended hydrophobic core are important for binding site selection by the Rnt1p dsRBD., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that binds to H/ACA RNAs specifically. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent from eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of Saccharomyces cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-β-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10 and RNA binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs expressed in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Telomerase is a ribonucleoprotein complex that replicates the 3' ends of linear chromosomes by successive additions of telomere repeat DNA. The telomerase holoenzyme contains two essential components for catalysis, a telomerase reverse transcriptase (TERT) and telomerase RNA (TER). The TER includes a template for telomere repeat synthesis as well as other domains required for function. We report the solution structure of the wild-type minimal conserved human TER pseudoknot refined with an extensive set of RDCs, and a detailed analysis of the effect of the bulge U177 on pseudoknot structure, dynamics analyzed by RDC and 13C relaxation measurements, and base pair stability. The overall structure of PKWT is highly similar to the previously reported DeltaU177 pseudoknot (PKDU) that has a deletion of a conserved bulge U important for catalytic activity. For direct comparison to PKWT, the structure of PKDU was re-refined with a comparable set of RDCs. Both pseudoknots contain a catalytically essential triple helix at the junction of the two stems, including two stem 1-loop 2 minor groove triples, a junction loop 1-loop 2 Hoogsteen base pair, and stem 2-loop 1 major groove U.A-U Watson-Crick-Hoogsteen triples located directly above the bulge U177. However, there are significant differences in the stabilities of base pairs near the bulge and the dynamics of some nucleotides. The stability of the base pairs in stem 2 surrounding the bulge U177 is greatly decreased, with the result that the Watson-Crick pairs in the triple helix begin to unfold before the Hoogsteen pairs, which may affect telomerase assembly and activity. The bulge U is positioned in the minor groove on the face opposite the triple helical interactions, and sterically blocks the A176 2'OH, which has recently been proposed to have a role in catalysis. The bulge U may serve as a hinge providing backbone flexibility in this region.

The Mg(2+)-dependent GAAA tetraloop interaction with its 11 nucleotide receptor is one of the most frequently occurring long-range tertiary interactions in RNAs. To explore conformational changes in the receptor during tetraloop docking, nitroxide spin labels were attached at each of four uridine bases, one at a time, within an RNA molecule containing the receptor sequence. In the presence of Mg2+ and the tetraloop, the electron paramagnetic resonance (EPR) spectrum of one of the labeled bases reflected a large increase in mobility, indicating unstacking of the base upon tetraloop docking. This provides direct evidence that base unstacking is an intrinsic feature of the solution tetraloop-receptor complex formed in the presence of Mg2+. Additional evidence suggests that in solution the bound receptor conformation is similar to that observed in the crystal structure of a group I intron ribozyme domain. In Mg2+ alone, a receptor conformation with an unstacked base was not detectable, suggesting that this conformation is of higher standard state free energy than that of the free receptor. This leads to the conclusion that the extensive RNA-RNA interactions observed in the crystal structure of the tetraloop-receptor complex provide larger interaction energy than the measured apparent affinity between the tetraloop and the free receptor. This is compatible with a high specificity of the tetraloop-receptor interaction.

Nucleolin is a 70 kDa multidomain protein involved in several steps of eukaryotic ribosome biogenesis. In vitro selection in combination with mutagenesis and structural analysis identified binding sites in pre-rRNA with the consensus (U/G)CCCG(A/G) in the context of a hairpin structure, the nucleolin recognition element (NRE). The central region of the protein contains four tandem RNA-binding domains (RBDs), of which the first two are responsible for the RNA-binding specificity and affinity for NREs. Here, we present the solution structure of the 28 kDa complex formed by the two N-terminal RNA-binding domains of nucleolin (RBD12) and a natural pre-rRNA target, b2NRE. The structure demonstrates that the sequence-specific recognition of the pre-rRNA NRE is achieved by intermolecular hydrogen bonds and stacking interactions involving mainly the beta-sheet surfaces of the two RBDs and the linker residues. A comparison with our previously determined NMR structure of RBD12 in complex with an in vitro selected RNA target, sNRE, shows that although the sequence-specific recognition of the loop consensus nucleotides is the same in the two complexes, they differ in several aspects. While the protein makes numerous specific contacts to the non-consensus nucleotides in the loop E motif (S-turn) in the upper part of the sNRE stem, nucleolin RBD12 contacts only consensus nucleotides in b2NRE. The absence of these upper stem contacts from the RBD12/b2NRE complex results in a much less stable complex, as demonstrated by kinetic analyses. The role of the loop E motif in high-affinity binding is supported by gel-shift analyses with a series of sNRE mutants. The less stable interaction of RBD12 with the natural RNA target is consistent with the proposed role of nucleolin as a chaperone that interacts transiently with pre-rRNA to prevent misfolding.

NHP6A is a non-sequence-specific DNA-binding protein from Saccharomyces cerevisiae which belongs to the HMGB protein family. Previously, we have solved the structure of NHP6A in the absence of DNA and modeled its interaction with DNA. Here, we present the refined solution structures of the NHP6A-DNA complex as well as the free 15bp DNA. Both the free and bound forms of the protein adopt the typical L-shaped HMGB domain fold. The DNA in the complex undergoes significant structural rearrangement from its free form while the protein shows smaller but significant conformational changes in the complex. Structural and mutational analysis as well as comparison of the complex with the free DNA provides insight into the factors that contribute to binding site selection and DNA deformations in the complex. Further insight into the amino acid determinants of DNA binding by HMGB domain proteins is given by a correlation study of NHP6A and 32 other HMGB domains belonging to both the DNA-sequence-specific and non-sequence-specific families of HMGB proteins. The resulting correlations can be rationalized by comparison of solved structures of HMGB proteins.

UBA domains are a commonly occurring sequence motif of approximately 45 amino acid residues that are found in diverse proteins involved in the ubiquitin/proteasome pathway, DNA excision-repair, and cell signaling via protein kinases. The human homologue of yeast Rad23A (HHR23A) is one example of a nucleotide excision-repair protein that contains both an internal and a C-terminal UBA domain. The solution structure of HHR23A UBA(2) showed that the domain forms a compact three-helix bundle. We report the structure of the internal UBA(1) domain of HHR23A. Comparison of the structures of UBA(1) and UBA(2) reveals that both form very similar folds and have a conserved large hydrophobic surface patch. The structural similarity between UBA(1) and UBA(2), in spite of their low level of sequence conservation, leads us to conclude that the structural variability of UBA domains in general is likely to be rather small. On the basis of the structural similarities as well as analysis of sequence conservation, we predict that this hydrophobic surface patch is a common protein-interacting surface present in diverse UBA domains. Furthermore, accumulating evidence that ubiquitin binds to UBA domains leads us to the prediction that the hydrophobic surface patch of UBA domains interacts with the hydrophobic surface on the five-stranded beta-sheet of ubiquitin. Detailed comparison of the structures of the two UBA domains, combined with previous mutagenesis studies, indicates that the binding site of HIV-1 Vpr on UBA(2) does not completely overlap the ubiquitin binding site., ((c) 2002 Elsevier Science Ltd.)

Nucleolin is an abundant nucleolar protein which is essential for ribosome biogenesis. The first two of its four tandem RNA-binding domains (RBD12) specifically recognize a stem-loop structure containing a conserved UCCCGA sequence in the loop called the nucleolin-recognition element (NRE). We have determined the structure of the consensus SELEX NRE (sNRE) by NMR spectroscopy. In both the free and bound RNA the top part of the stem forms a loop E (or S-turn) motif. In the absence of protein, the structure of the hairpin loop is not well defined due to conformational heterogeneity, and appears to be in equilibrium between two families of conformations. Titrations of RBD1, RBD2, and RBD12 with the sNRE show that specific binding requires RBD12. In complex with RBD12, the hairpin loop interacts specifically with the protein and adopts a well-defined structure which shares some of the features of the free form. The loop E motif also has specific interactions with the protein. Implications of these findings for the mechanism of recognition of RNA structures by modular proteins are discussed., (Copyright 2001 Academic Press.)

Nucleolin is an abundant 70 kDa nucleolar protein involved in many aspects of ribosomal RNA biogenesis. The central region of nucleolin contains four tandem consensus RNA-binding domains (RBD). The two most N-terminal domains (RBD12) bind with nanomolar affinity to an RNA stem-loop containing the consensus sequence UCCCGA in the loop. We have determined the solution structure of nucleolin RBD12 in its free form and have studied its interaction with a 22 nt RNA stem-loop using multidimensional NMR spectroscopy. The two RBDs adopt the expected beta alpha beta beta alpha beta fold, but the position of the beta 2 strand in both domains differs from what was predicted from sequence alignments. RBD1 and RBD2 are significantly different from each others and this is likely important in their sequence specific recognition of the RNA. RBD1 has a longer alpha-helix 1 and a shorter beta 2-beta 3 loop than RBD2, and differs from most other RBDs in these respects. The two RBDs are separated by a 12 amino acid flexible linker and do not interact with one another in the free protein. This linker becomes ordered when RBD12 binds to the RNA. Analysis of the observed NOEs between the protein and the RNA indicates that both RBDs interact with the RNA loop via their beta-sheet. Each domain binds residues on one side of the loop; specifically, RBD2 contacts the 5' side and RBD1 contacts the 3'., (Copyright 2000 Academic Press.)

Monovalent cation binding sites on nucleic acids in solution can be localized using the isotopically labeled ammonium ion (15NH4+) as a probe in high resolution NMR spectroscopy experiments. The application of this technique to a series of DNA duplexes reveals a preference for the binding of ammonium cations in the minor groove of A-tract sequences. These results are consistent with a recent report which indicates that some solvent electron densities previously identified as water molecules in DNA X-ray crystal structures are partially occupied by sodium ions. The sequence-specific nature of monovalent cation binding sites demonstrated here for A-tract DNA provides an explanation for the origin of sequence-directed bending., (Copyright 1999 Academic Press.)

Guanine quartets are readily formed by guanine nucleotides and guanine-rich oligonucleotides in the presence of certain monovalent and divalent cations. The quadruplexes composed of these quartets are of interest for their potential roles in vivo, their relatively frequent appearance in oligonucleotides derived from in vitro selection, and their inhibition of template directed RNA polymerization under proposed prebiotic conditions. The requirement of cation coordination for the stabilization of G quartets makes understanding cation-quadruplex interactions an essential step towards a complete understanding of G quadruplex formation. We have used 15NH4+ as a probe of cation coordination by the four G quartets of the DNA bimolecular quadruplex [d(G4T4G4)]2, formed from oligonucleotides with the repeat sequence found in Oxytricha nova telomeres. 1H and 15N heteronuclear NMR spectroscopy has allowed the direct localization of monovalent cation binding sites in the solution state and the analysis of cation movement between the binding sites. These experiments show that [d(G4T4G4)]2 coordinates three ammonium ions, one in each of two symmetry related sites and one on the axis of symmetry of the dimeric molecule. The NH4+ move along the central axis of the quadruplex between these sites and the solution, reminiscent of an ion channel. The residence time of the central ion is determined to be 250 ms. The 15NH4+ is shown to be a valuable probe of monovalent cation binding sites and dynamics., (Copyright 1999 Academic Press.)

The solution structure of the ATP-binding RNA aptamer has recently been determined by NMR spectroscopy. The three-dimensional fold of the molecule is determined to a large extent by stacking and hydrogen bond interactions. In the course of the structure determination it was discovered that several highly conserved nucleotides in the binding pocket can be substituted while retaining binding under NMR conditions. These surprising findings allow a closer look at the interactions that determine stability and specificity of the aptamer as well as local structural features of the molecule. The binding properties of ATP binder mutants and modified ligand molecules are explored using NMR spectroscopy, column binding studies and molecular modeling. We present additional evidence and new insights regarding the network of hydrogen bonds that defines the structure and determines stability and specificity of the aptamer., (Copyright 1997 Academic Press Limited.)

The solution structure of the highly conserved UGAA tetraloop found at the 3' end of eukaryotic 16 S-like ribosomal RNA has been solved by nuclear magnetic resonance spectroscopy in the form of the 12 nucleotide hairpin 5'-GGUG[UGAA]CACC. The UGAA tetraloop displays a novel fold. The backbone turn occurs between the G and the third A in the loop, with the U and G in a 5' stack and the As in a 3' stacking arrangement. The loop is closed by a U-A mismatch in which the O2, 2'OH, and O4' groups of the U are within hydrogen bonding distance of the amino group of the A. The tetraloop does not make a uridine-turn, even though its sequence is identical to a U-turn found within the anticodon loop of tRNA(Phe). The hydrogen bonding pattern in the tetraloop provides insight into the function of base modifications found in vivo within this portion of 16 S-like rRNA.

The HIV-1 Rev responsive element (RRE) high-affinity binding site was studied by homonuclear and heteronuclear NMR. Two Rev binding element (RBE) RNA oligonucleotides were used as model systems in this study: RBE3, which contains the wild-type Rev high-affinity binding site, and RBE3-A which is identical except for the deletion of a bulged A. The temperature dependence of the two-dimensional spectra of the free RNAs indicates that at lower temperatures more than one conformation is present. However, at higher temperatures a single conformation predominates. Model structures of RBE3 and RBE3-A as well as the RBE3-A complexed with a peptide derived from the RNA binding domain of HIV-1 Rev, were calculated using NMR-derived restraints. The Rev high-affinity binding site of the HIV-1 RRE contains a structured internal loop with two purine-purine base-pairs and an extrahelical U. Comparison of the free and bound RNA structures reveals that upon peptide binding there is a distinct change in the backbone at G24, which is involved in a G-G base-pair. In the free RNA, G24 is in the syn conformation, and the backbone is in a relatively normal configuration, antiparallel to the other strand. In the bound RNA, the backbone at G24 has flipped over so that it is parallel to the other strand. G24 in the bound RNA still forms a base-pair with G6, but is now in the anti conformation.

We have used NMR spectroscopy to study a pyrimidine-purine-pyrimidine DNA triplex containing a non-natural base, 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamido)phenylimidazole (D3), in the third strand. The D3 base has been previously shown to specifically recognize T-A and C-G base-pairs via intercalation on the 3' side (with respect to the purine strand) of the target base pair, instead of forming sequence-specific hydrogen bonds. 1H resonance assignments have been made for the D3 base and most of the non-loop portion of the triplex. The solution structure of the triplex was calculated using restrained molecular dynamics and complete relaxation matrix refinement. The duplex portion of the triplex has an over-all helical structure that is more similar to B-DNA than to A-DNA. The three aromatic rings of the D3 base stack on the bases of all three strands and mimic a triplet. The conformation of the D3 base and its sequence specificity are discussed.

The thrombin-binding aptamer d(GGTTGGTGTGGTTGG) is one of a family of DNA oligonucleotides that were identified by in vitro selection to bind specifically and with high affinity to thrombin. Two groups independently determined the tertiary structure in solution by NMR and at about the same time, the X-ray crystal structure of the aptamer in complex with thrombin was reported. In all cases, the thrombin-binding aptamer was found to fold into a structure containing two planar guanine quartets as its core. The NMR and crystal structures, however, have fundamentally different folding patterns owing to differences in the way these central bases are connected. We discuss the distinctions between the refined crystal and solution structures and show that the NMR model is consistent with the X-ray diffraction data.

The DNA oligonucleotide d(GGTTGGTGTGGTTGG) (thrombin aptamer) binds to thrombin and inhibits its enzymatic activity in the chain of reactions that lead to blood clotting. Two-dimensional 1H NMR studies indicate that the oligonucleotide forms a folded structure in solution, composed of two guanine quartets connected by two T-T loops spanning the narrow grooves at one end and a T-G-T loop spanning a wide groove at the other end. We present the assignment strategy used, methods for the structure determination, and the refined three-dimensional structure of the thrombin aptamer. The initial structures were generated by metric matrix distance geometry using distance and dihedral bond angle constraints from NOE and coupling constants, respectively, and refined by restrained molecular dynamics and direct NOE refinement. Knowledge of the three-dimensional structure of this thrombin aptamer may be relevant for the design of improved thrombin-inhibiting anti-coagulants with similar structural motifs.

Two-dimensional 1H n.m.r. spectroscopy has been used to study the 31-base DNA oligonucleotide 5'-dAGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3', which folds to form a stable intramolecular triplex in solution at acidic pH. This structure is considerably more difficult to assign than short B-DNA duplexes and requires new assignment methods. The assignment strategy and assignments of almost all of the exchangeable and nonexchangeable resonances are presented. Seven base triplets and one Watson-Crick base-pair form the core of the structure and are connected by a four C and four T loop at either end. The second pyrimidine "strand" (bases 24 to 31) in this intramolecular pyrimidine-purine-pyrimidine triplex binds via Hoogsteen base-pairs in the major groove and is parallel to the purine "strand" (bases 1 to 8). Analysis of the sugar puckers reveals that, contrary to widely accepted belief, the triplex sugars are not predominantly in the N-type (close to C3'-endo) conformation. Except for some of the C nucleotides, all sugars are predominantly S-type (close to C2'-endo). Thus, the duplex DNA does not assume N-type sugar conformations to accommodate a third strand in the major groove. A preliminary model of the triplex structure is presented.