Biomedical subjects

Juli Feigon

Publications and source records attributed to Juli Feigon.

At least 19 recordsLinked to original sources

PubMed2006

Structural study of elements of Tetrahymena telomerase RNA stem-loop IV domain important for function.

Tetrahymena telomerase RNA (TER) contains several regions in addition to the template that are important for function. Central among these is the stem-loop IV domain, which is involved in both catalysis and RNP assembly, and includes binding sites for both the holoenzyme assembly protein p65 and telomerase reverse transcriptase (TERT). Stem-loop IV contains two regions with high evolutionary sequence conservation: a central GA bulge between helices, and a terminal loop. We solved the solution structure of loop IV and modeled the structure of the helical region containing the GA bulge, using NMR and residual dipolar couplings. The central GA bulge with flanking C-G base pairs induces a approximately 50 degrees semi-rigid bend in the helix. Loop IV is highly structured, and contains a conserved C-U base pair at the top of the helical stem. Analysis of new and previous biochemical data in light of the structure provides a rationale for some of the sequence conservation in this region of TER. The results suggest that during holoenzyme assembly the protein p65 recognizes a bend in stem IV, and this binding to central stem IV helps to position the structured loop IV for interaction with TERT and other region(s) of TER.

Animals↗

PubMed2006

Structure and function of telomerase RNA.

Maintenance of telomeres by the enzyme telomerase is essential for genomic stability and cell viability in ciliates, vertebrates and yeast. The minimal components of telomerase required for catalytic activity are the telomerase reverse transcriptase (TERT) protein and the template-containing telomerase RNA (TER). Recent studies have afforded significant advances in the biophysical characterization of telomerase RNAs from various species. The first TER structures have been reported, for regions of the catalytically essential pseudoknot and CR4/CR5 domains of human TER, and provide a structural basis for interpretation of mutational and biochemical data. The domains and interactions of the Tetrahymena thermophila telomerase holoenzyme RNA and protein components have been further characterized biochemically, and structures of the TER template boundary element and the N-terminal domain of T. thermophila TERT have been determined. Phylogenetic and biochemical analyses of yeast TERs have revealed core structural elements in common with ciliates and vertebrates, and the minimal domains required for function in vivo.

Animals↗

PubMed2006

Structure of the Tetrahymena thermophila telomerase RNA helix II template boundary element.

Telomere addition by telomerase requires an internal templating sequence located in the RNA subunit of telomerase. The correct boundary definition of this template sequence is essential for the proper addition of the nucleotide repeats. Incorporation of incorrect telomeric repeats onto the ends of chromosomes has been shown to induce chromosomal instability in ciliate, yeast and human cells. A 5' template boundary defining element (TBE) has been identified in human, yeast and ciliate telomerase RNAs. Here, we report the solution structure of the TBE element (helix II) from Tetrahymena thermophila telomerase RNA. Our results indicate that helix II and its capping pentaloop form a well-defined structure including unpaired, stacked adenine nucleotides in the stem and an unusual syn adenine nucleotide in the loop. A comparison of the T.thermophila helix II pentaloop with a pentaloop of the same sequence found in the 23S rRNA of the Haloarcula marismortui ribosome suggests possible RNA and/or protein interactions for the helix II loop within the Tetrahymena telomerase holoenzyme.

Animals↗

PubMed2006

Structural study of the H/ACA snoRNP components Nop10p and the 3' hairpin of U65 snoRNA.

The H/ACA small nucleolar ribonucleoprotein (snoRNP) complexes guide the modification of uridine to pseudouridine at conserved sites in rRNA. The H/ACA snoRNPs each comprise a target-site-specific snoRNA and four core proteins, Nop10p, Nhp2p, Gar1p, and the pseudouridine synthase, Cbf5p, in yeast. The secondary structure of the H/ACA snoRNAs includes two hairpins that each contain a large internal loop (the pseudouridylation pocket), one or both of which are partially complementary to the target RNA(s). We have determined the solution structure of an RNA hairpin derived from the human U65 box H/ACA snoRNA including the pseudouridylation pocket and adjacent stems, providing the first three-dimensional structural information on these H/ACA snoRNAs. We have also determined the structure of Nop10p and investigated its interaction with RNA using NMR spectroscopy. Nop10p contains a structurally well-defined N-terminal region composed of a beta-hairpin, and the rest of the protein lacks a globular structure. Chemical shift mapping of the interaction of RNA constructs of U65 box H/ACA 3' hairpin with Nop10p shows that the beta-hairpin binds weakly but specifically to RNA. The unstructured region of Nop10p likely interacts with Cbf5p.

Amino Acid Sequence↗

PubMed2005

Characterization of the cation and temperature dependence of DNA quadruplex hydrogen bond properties using high-resolution NMR.

Variations in the hydrogen bond network of the Oxy-1.5 DNA guanine quadruplex have been monitored by trans-H-bond scalar couplings, (h2)J(N2N7), for Na(+)-, K(+)-, and NH(4)(+)-bound forms over a temperature range from 5 to 55 degrees C. The variations in (h2)J(N2N7) couplings exhibit an overall trend of Na(+) > K(+) > NH(4)(+) and correlate with the different cation positions and N2-H2...N7 H-bond lengths in the respective structures. A global weakening of the (h2)J(N2N7) couplings with increasing temperature for the three DNA quadruplex species is accompanied by a global increase of the acceptor (15)N7 chemical shifts. Above 35 degrees C, spectral heterogeneity indicates thermal denaturation for the Na(+)-bound form, whereas spectral homogeneity persists up to 55 degrees C for the K(+)- and NH(4)(+)-coordinated forms. The average relative change of the (h2)J(N2N7) couplings amounts to approximately 0.8 x 10(-3)/K and is thus considerably smaller than respective values reported for nucleic acid duplexes. The significantly higher thermal stability of H-bond geometries in the DNA quadruplexes can be rationalized by their cation coordination of the G-quartets and the extensive H-bond network between the four strands. A detailed analysis of individual (h2)J(N2N7) couplings reveals that the 5' strand end, comprising base pairs G1-G9* and G4*-G1, is the most thermolabile region of the DNA quadruplex in all three cation-bound forms.

Cations↗

PubMed2005

Site-directed spin labeling studies reveal solution conformational changes in a GAAA tetraloop receptor upon Mg(2+)-dependent docking of a GAAA tetraloop.

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.

Base Sequence↗

PubMed2005

Biochemical and genomic analysis of substrate recognition by the double-stranded RNA binding domain of yeast RNase III.

Members of the RNase III family of double-stranded RNA (dsRNA) endonucleases are important enzymes of RNA metabolism in eukaryotic cells. Rnt1p is the only known member of the RNase III family of endonucleases in Saccharomyces cerevisiae. Previous studies have shown that Rnt1p cleaves dsRNA capped by a conserved AGNN tetraloop motif, which is a major determinant for Rnt1p binding and cleavage. The solution structure of the dsRNA-binding domain (dsRBD) of Rnt1p bound to a cognate RNA substrate revealed the structural basis for binding of the conserved tetraloop motif by alpha-helix 1 of the dsRBD. In this study, we have analyzed extensively the effects of mutations of helix 1 residues that contact the RNA. We show, using microarray analysis, that mutations of these amino acids induce substrate-specific processing defects in vivo. Cleavage kinetics and binding studies show that these mutations affect RNA cleavage and binding in vitro to different extents and suggest a function for some specific amino acids of the dsRBD in the catalytic positioning of the enzyme. Moreover, we show that 2'-hydroxyl groups of nucleotides of the tetraloop or adjacent base pairs predicted to interact with residues of alpha-helix 1 are important for Rnt1p cleavage in vitro. This study underscores the importance of a few amino acid contacts for positioning of a dsRBD onto its RNA target, and implicates the specific orientation of helix 1 on the RNA for proper positioning of the catalytic domain.

Base Sequence↗

PubMed2005

Structure of the human telomerase RNA pseudoknot reveals conserved tertiary interactions essential for function.

Human telomerase contains a 451 nt RNA (hTR) and several proteins, including a specialized reverse transcriptase (hTERT). The 5' half of hTR comprises the pseudoknot (core) domain, which includes the RNA template for telomere synthesis and a highly conserved pseudoknot that is required for telomerase activity. The solution structure of this essential pseudoknot, presented here, reveals an extended triple helix surrounding the helical junction. The network of tertiary interactions explains the phylogenetic sequence conservation and existing human and mouse TR functional studies as well as mutations linked to disease. Thermodynamic stability, dimerization potential, and telomerase activity of mutant RNAs that alter the tertiary contacts were investigated. Telomerase activity is strongly correlated with tertiary structure stability, whereas there is no correlation with dimerization potential of the pseudoknot. These studies reveal that a conserved pseudoknot tertiary structure is required for telomerase activity.

Animals↗

PubMed2005

Structure determination of protein/RNA complexes by NMR.

Structure determination of protein?RNA complexes in solution provides unique insights into factors that are involved in protein/RNA recognition. Here, we review the methodology used in our laboratory to overcome the challenges of protein?RNA structure determination by nuclear magnetic resonance (NMR). We use as two examples complexes recently solved in our laboratory, the nucleolin RBD12/b2NRE and Rnt1p dsRBD/snR47h complexes. Topics covered are protein and RNA preparation, complex formation, identification of the protein/RNA interface, protein and RNA resonance assignment, intermolecular NOE assignment, and structure calculation and analysis.

Magnetic Resonance Spectroscopy↗

PubMed2004

Contributions of the RNA-binding and linker domains and RNA structure to the specificity and affinity of the nucleolin RBD12/NRE interaction.

Nucleolin is a multidomain phosphoprotein involved in ribosome biogenesis. In vitro selection and binding studies with pre-rRNA fragments have shown that the first two RNA-binding domains (RBDs) in nucleolin (RBD12) recognize the consensus sequence (U/G)CCCG(A/G) in the context of a stem-loop structure (nucleolin-recognition element = NRE). Structural studies of nucleolin RBD12 in complex with an in vitro selected NRE (sNRE) and a natural pre-rRNA NRE (b2NRE) have revealed that sequence-specific binding of the consensus NRE is achieved in a similar manner in both complexes using residues in both RBDs as well as the linker connecting them. Using fluorescence anisotropy (FA) and nuclear magnetic resonance (NMR), we demonstrate the importance of the linker for NRE affinity by showing that only the individual RBDs with the linker attached retain the ability to specifically bind, albeit weakly, to sNRE and b2NRE. Binding of RBD1 and RBD2 to the NREs in trans is not detected even when one of the RBDs has the linker attached, which suggests that the linker also contributes to the affinity by tethering the two RBDs. To determine if binding of nucleolin RBD12 to natural NREs is dependent on a specific RNA stem-loop structure, as was the case for the sNRE, we conducted FA and NMR binding assays with nucleolin RBD12 and a single-stranded NRE. The results show that nucleolin RBD12 sequence-specifically binds a single-stranded NRE with an affinity similar to that for b2NRE, indicating that a stem-loop structure is not required for the nucleolin RBD12/pre-rRNA NRE interaction.

Amino Acid Sequence↗

PubMed2004

Structural basis for recognition of the AGNN tetraloop RNA fold by the double-stranded RNA-binding domain of Rnt1p RNase III.

Specific recognition of double-stranded RNA (dsRNA) by dsRNA-binding domains (dsRBDs) is involved in a large number of biological and regulatory processes. Although structures of dsRBDs in complex with dsRNA have revealed how they can bind to dsRNA in general, these do not explain how a dsRBD can recognize specific RNAs. Rnt1p, a member of the RNase III family of dsRNA endonucleases, is a key component of the Saccharomyces cerevisiae RNA-processing machinery. The Rnt1p dsRBD has been implicated in targeting this endonuclease to its RNA substrates, by recognizing hairpins closed by AGNN tetraloops. We report the solution structure of Rnt1p dsRBD complexed to the 5' terminal hairpin of one of its small nucleolar RNA substrates, the snR47 precursor. The conserved AGNN tetraloop fold is retained in the protein-RNA complex. The dsRBD contacts the RNA at successive minor, major, and tetraloop minor grooves on one face of the helix. Surprisingly, neither the universally conserved G nor the highly conserved A are recognized by specific hydrogen bonds to the bases. Rather, the N-terminal helix fits snugly into the minor groove of the RNA tetraloop and top of the stem, interacting in a non-sequence-specific manner with the sugar-phosphate backbone and the two nonconserved tetraloop bases. Mutational analysis of residues that contact the tetraloop region show that they are functionally important for RNA processing in the context of the entire protein in vivo. These results show how a single dsRBD can convey specificity for particular RNA targets, by structure specific recognition of a conserved tetraloop fold.

Amino Acid Sequence↗

PubMed2004

Solution structure of the complex formed by the two N-terminal RNA-binding domains of nucleolin and a pre-rRNA target.

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.

Animals↗

PubMed2004

DNA A-tract bending in three dimensions: solving the dA4T4 vs. dT4A4 conundrum.

DNA A-tracts have been defined as four or more consecutive A.T base pairs without a TpA step. When inserted in phase with the DNA helical repeat, bending is manifested macroscopically as anomalous migration on polyacrylamide gels, first observed >20 years ago. An unsolved conundrum is why DNA containing in-phase A-tract repeats of A(4)T(4) are bent, whereas T(4)A(4) is straight. We have determined the solution structures of the DNA duplexes formed by d(GCAAAATTTTGC) [A4T4] and d(CGTTTTAAAACG) [T4A4] with NH(4)(+) counterions by using NMR spectroscopy, including refinement with residual dipolar couplings. Analysis of the structures shows that the ApT step has a large negative roll, resulting in a local bend toward the minor groove, whereas the TpA step has a positive roll and locally bends toward the major groove. For A4T4, this bend is nearly in phase with bends at the two A-tract junctions, resulting in an overall bend toward the minor groove of the A-tract, whereas for T4A4, the bends oppose each other, resulting in a relatively straight helix. NMR-based structural modeling of d(CAAAATTTTG)(15) and d(GTTTTAAAAC)(15) reveals that the former forms a left-handed superhelix with a diameter of approximately 110 A and pitch of 80 A, similar to DNA in the nucleosome, whereas the latter has a gentle writhe with a pitch of >250 A and diameter of approximately 50 A. Results of gel electrophoretic mobility studies are consistent with the higher-order structure of the DNA and furthermore depend on the nature of the monovalent cation present in the running buffer.

Crystallization↗

PubMed2004

Specificity of the interaction between ubiquitin-associated domains and ubiquitin.

Ubiquitin-associated (UBA) domains are found in a large number of proteins with diverse functions involved in ubiquitination, DNA repair, and signaling pathways. Recent studies have shown that several UBA domain proteins interact with ubiquitin (Ub), specifically p62, the phosphotyrosine-independent ligand of the SH2 domain of p56(lck); HHR23A, a human nucleotide excision repair protein; and DDI1, another damage-inducible protein. NMR chemical shift mapping reveals that Ub binds specifically but weakly to a conserved hydrophobic epitope on HHR23A UBA(1) and UBA(2) and that the UBA domains bind on the hydrophobic patch on the surface of the five-stranded beta-sheet of Ub. Models of the UBA(1)-Ub and UBA(2)-Ub complexes obtained from de novo docking reveal different orientations of the UBA domains on the Ub surface compared with those obtained by homology modeling with the related CUE domains, which also bind Ub. Our results suggest that UBA domains may interact with Ub as well as other proteins in more than one way while utilizing the same binding surface.

Amino Acid Sequence↗

PubMed2004

New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-protein complexes.

NMR spectra of large RNAs are difficult to assign because of extensive spectral overlap and unfavorable relaxation properties. Here we present a new approach to facilitate assignment of RNA spectra using a suite of four 2D-filtered/edited NOESY experiments in combination with base-type-specific isotopically labeled RNA. The filtering method was developed for use in 3D filtered NOESY experiments (Zwahlen et al., 1997), but the 2D versions are both more sensitive and easier to interpret for larger RNAs than their 3D counterparts. These experiments are also useful for identifying intermolecular NOEs in RNA-protein complexes. Applications to NOE assignment of larger RNAs and an RNA-protein complex are presented.

Macromolecular Substances↗

PubMed2004

Structure of the XPC binding domain of hHR23A reveals hydrophobic patches for protein interaction.

Rad23 proteins are involved both in the ubiquitin-proteasome pathway and in nucleotide excision repair (NER), but the relationship between these two pathways is not yet understood. The two human homologs of Rad23, hHR23A and B, are functionally redundant in NER and interact with xeroderma pigmentosum complementation group C (XPC) protein. The XPC-hHR23 complex is responsible for the specific recognition of damaged DNA, which is an early step in NER. The interaction of the XPC binding domain (XPCB) of hHR23A/B with XPC protein has been shown to be important for its optimal function in NER. We have determined the solution structure of XPCB of hHR23A. The domain consists of five amphipathic helices and reveals hydrophobic patches on the otherwise highly hydrophilic domain surface. The patches are predicted to be involved in interaction with XPC. The XPCB domain has limited sequence homology with any proteins outside of the Rad23 family except for sacsin, a protein involved in spastic ataxia of Charlevoix-Saguenay, which contains a domain with 35% sequence identity.

Amino Acid Sequence↗

PubMed2003

Solution structures of stem-loop RNAs that bind to the two N-terminal RNA-binding domains of nucleolin.

Nucleolin, a multi-domain protein involved in ribosome biogenesis, has been shown to bind the consensus sequence (U/G)CCCG(A/G) in the context of a hairpin loop structure (nucleolin recognition element; NRE). Previous studies have shown that the first two RNA-binding domains in nucleolin (RBD12) are responsible for the interaction with the in vitro selected NRE (sNRE). We have previously reported the structures of nucleolin RBD12, sNRE and nucleolin RBD12-sNRE complex. A comparison of free and bound sNRE shows that the NRE loop becomes structured upon binding. From this observation, we hypothesized that the disordered hairpin loop of sNRE facilitates conformational rearrangements when the protein binds. Here, we show that nucleolin RBD12 is also sufficient for sequence- specific binding of two NRE sequences found in pre-rRNA, b1NRE and b2NRE. Structural investigations of the free NREs using NMR spectroscopy show that the b1NRE loop is conformationally heterogeneous, while the b2NRE loop is structured. The b2NRE forms a hairpin capped by a YNMG-like tetraloop. Comparison of the chemical shifts of sNRE and b2NRE in complex with nucleolin RBD12 suggests that the NRE consensus nucleotides adopt a similar conformation. These results show that a disordered NRE consensus sequence is not a prerequisite for nucleolin RBD12 binding.

Base Sequence↗

PubMed2003

Structural determinants for the binding of ubiquitin-like domains to the proteasome.

HHR23A, a protein implicated in nucleotide excision repair, belongs to a class of proteins containing both a ubiquitin-like (Ubl) domain and one or more ubiquitin-associated (UBA) domains, suggesting a role in the ubiquitin-proteasome pathway as well. The Ubl domain binds with high affinity to the second ubiquitin-interacting motif (UIM) of the S5a subunit of the proteasome. Here we present the solution structures of the HHR23A Ubl domain, the second UIM of S5a (UIM-2), and the Ubl:S5a-UIM-2 complex. The HHR23A Ubl domain is structurally similar to ubiquitin. The S5a UIM forms an alpha-helix with an unexpected hairpin loop that contributes to the binding interface with Ubl. The molecular determinants of the Ubl-proteasome interaction are revealed by analysis of the structures, chemical shift mapping, mutant binding studies and sequence conservation.

Amino Acid Sequence↗