Structure of S. pombe telomerase protein Pof8 C-terminal domain is an xRRM conserved among LARP7 proteins.

La-related proteins 7 (LARP7) are a class of RNA chaperones that bind the 3' ends of RNA and are constitutively associated with their specific target RNAs. In metazoa, Larp7 binds to the long non-coding 7SK RNA as a core component of the 7SK RNP, a major regulator of eukaryotic transcription. In the ciliate Tetrahymena the LARP7 protein p65 is a component of telomerase, an essential ribonucleoprotein complex that maintains the telomeric DNA at eukaryotic chromosome ends. p65 is important for the ordered assembly of telomerase RNA (TER) with telomerase reverse transcriptase. Unexpectedly, Schizosaccharomyces pombe Pof8 was recently identified as a LARP7 protein and a core component of fission yeast telomerase essential for biogenesis. LARP7 proteins have a conserved N-terminal La motif and RRM1 (La module) and C-terminal RRM2 with specific RNA substrate recognition attributed to RRM2, first structurally characterized in p65 as an atypical RRM named xRRM. Here we present the X-ray crystal structure and NMR studies of S. pombe Pof8 RRM2. Sequence and structure comparison of Pof8 RRM2 to p65 and human Larp7 xRRMs reveals conserved features for RNA binding with the main variability in the length of the non-canonical helix α3. This study shows that Pof8 has conserved xRRM features, providing insight into TER recognition and the defining characteristics of the xRRM.

Structurally conserved five nucleotide bulge determines the overall topology of the core domain of human telomerase RNA.

Telomerase is a unique ribonucleoprotein complex that catalyzes the addition of telomeric DNA repeats onto the 3' ends of linear chromosomes. All vertebrate telomerase RNAs contain a catalytically essential core domain that includes the template and a pseudoknot with extended helical subdomains. Within these helical regions is an asymmetric 5-nt internal bulge loop (J2a/b) flanked by helices (P2a and P2b) that is highly conserved in its location but not sequence. NMR structure determination reveals that J2a/b forms a defined S-shape and creates an ∼90 ° bend with a surprisingly low twist (∼10 °) between the flanking helices. A search of RNA structures revealed only one other example of a 5-nt bulge, from hepatitis C virus internal ribosome entry site, with a different sequence but the same structure. J2a/b is intrinsically flexible but the interhelical motions across the loop are remarkably restricted. Nucleotide substitutions in J2a/b that affect the bend angle, direction, and interhelical dynamics are correlated with telomerase activity. Based on the structures of P2ab (J2a/b and flanking helices), the conserved region of the pseudoknot (P2b/P3, previously determined) and the remaining helical segment (P2a.1-J2a.1 refined using residual dipolar couplings and the modeling program MC-Sym) we have calculated an NMR-based model of the full-length pseudoknot. The model and dynamics analysis show that J2a/b serves as a dominant structural and dynamical element in defining the overall topology of the core domain, and suggest that interhelical motions in P2ab facilitate nucleotide addition along the template and template translocation.

Comparison of solution and crystal structures of preQ1 riboswitch reveals calcium-induced changes in conformation and dynamics.

Riboswitches regulate gene expression via specific recognition of cognate metabolites by their aptamer domains, which fold into stable conformations upon ligand binding. However, the recently reported solution and crystal structures of the Bacillus subtilis preQ(1) riboswitch aptamer show small but significant differences, suggesting that there may be conformational heterogeneity in the ligand-bound state. We present a structural and dynamic characterization of this aptamer by solution NMR spectroscopy. The aptamer-preQ(1) complex is intrinsically flexible in solution, with two regions that undergo motions on different time scales. Three residues move in concert on the micro-to-millisecond time scale and may serve as the lid of the preQ(1)-binding pocket. Several Ca(2+) ions are present in the crystal structure, one of which binds with an affinity of 47 ± 2 µM in solution to a site that is formed only upon ligand binding. Addition of Ca(2+) to the aptamer-preQ(1) complex in solution results in conformational changes that account for the differences between the solution and crystal structures. Remarkably, the Ca(2+) ions present in the crystal structure, which were proposed to be important for folding and ligand recognition, are not required for either in solution.

Compensating increases in protein backbone flexibility occur when the Dead ringer AT-rich interaction domain (ARID) binds DNA: a nitrogen-15 relaxation study.

AT-rich interaction domains (ARIDs) are found in a large number of eukaryotic transcription factors that regulate cell proliferation, differentiation, and development. Previously we elucidated how ARIDs recognize DNA by determining the solution structure of the Drosophila melanogaster Dead ringer protein in both its DNA-free and -bound states. In order to quantitatively determine how ARIDs alter their mobility to recognize DNA, we have measured the relaxation parameters of the backbone nitrogen-15 nuclei of Dead ringer in its free and bound forms, and interpreted these data using the model-free approach. We show that Dead ringer undergoes significant changes in its mobility upon binding, with residues in the loop connecting helices H5 and H6 becoming immobilized in the major groove and contacts to the minor groove slowing down the motion of residues at the C terminus. A DNA-induced rotation and displacement of the N-terminal subdomain of the protein increases the mobility of helix H1 located distal to the DNA interface and may partially negate the entropic cost of immobilizing interfacial residues. Elevated motions on the micro- to millisecond timescale in the N-terminal domain prior to DNA binding appear to foreshadow the DNA-induced conformation change.

Solution structure and dynamics of the wild-type pseudoknot of human telomerase RNA.

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.

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.

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.