NMR structure of a ribosomal RNA hairpin containing a conserved CUCAA pentaloop (original) (raw)
Related papers
Biochemistry, 2001
The binding region of the Escherichia coli S2 ribosomal protein contains a conserved UUAAGU hairpin loop. The structure of the hairpin formed by the oligomer r(GCGU4U5A6A7G8U9CGCA), which has an r(UUAAGU) hairpin loop, was determined by NMR and molecular modeling techniques as part of a study aimed at characterizing the structure and thermodynamics of RNA hairpin loops. Thermodynamic data obtained from melting curves for this RNA oligomer show that it forms a hairpin in solution with the following parameters: ∆H°)-42.8 (2.2 kcal/mol, ∆S°)-127.6 (6.5 eu, and ∆G°3 7)-3.3 (0.2 kcal/mol. Two-dimensional NOESY WATERGATE spectra show an NOE between U imino protons, which suggests that U4 and U9 form a hydrogen bonded U‚U pair. The U5(H2′) proton shows NOEs to both the A6(H8) proton and the A7(H8) proton, which is consistent with formation of a "U" turn between nucleotides U5 and A6. An NOE between the A7(H2) proton and the U9(H4′) proton shows the proximity of the A7 base to the U9 sugar, which is consistent with the structure determined for the six-nucleotide loop. In addition to having a hydrogen-bonded U‚U pair as the first mismatch and a U turn, the r(UUAAGU) loop has the G8 base protruding into the solvent. The solution structure of the r(UUAAGU) loop is essentially identical to the structure of an identical loop found in the crystal structure of the 30S ribosomal subunit where the guanine in the loop is involved in tertiary interactions with RNA bases from adjacent regions [Wimberly, B.
Journal of Molecular Biology, 2001
The solution structure of the conserved 690 hairpin from Escherichia coli 16 S rRNA was determined by NMR spectroscopy. The 690 loop is located at the surface of the 30 S subunit in the platform region and has been implicated in interactions with P-site bound tRNA, E-site mRNA, S11 binding, IF3 binding, and in RNA-RNA interactions with the 790 loop of 16 S rRNA and domain IV of 23 S rRNA. The structure reveals a novel sheared type G690 ÁU697 base-pair with a single hydrogen bond from the G690 amino to U697-04. G691 and A696 also form a sheared pair and U692 forms a U-turn with an H-bond to the A695 non-bridging phosphate oxygen. The sheared pairs and U-turn result in the continuous single-stranded stacking of ®ve residues from 6693 to U697 with their Watson-Crick functional groups exposed in the minor groove. The overall fold of the 690 hairpin is similar to the anticodon loop of tRNA. The structure provides an explanation for chemical protection patterns in the loop upon interaction with tRNA, the 50 S subunit, and S11. In vivo genetic studies demonstrate the functional importance of the motifs observed in the solution structure of the 690 hairpin.
Proton NMR and Structural Features of a 24Nucleotide RNA Hairpin
Biochemistry, 1995
The three-dimensional conformation of a 24-nucleotide variant of the RNA binding sequence for the coat protein of bacteriophage R17 has been analyzed using NMK. molecular dynamics, and energy minimization. The imino proton spectrum is consistent with base pairing requirements for coat protein binding known from biochemical studies. All 185 of the nonexchangeable protons were assigned using a variety of homonuclear 2D and 3D NMR methods. Measurements of nuclear Overhauser enhancements and two-quantum correlations were made at 500 MHz. New procedures were developed to characterize as many resonances as possible, including deconvolution and path analysis methods. An average of 21 &stance constraints per residue were used in molecular dynamics calculations to obtain preliminary folded structures for residues 3-21. The unpaired A8 residue is stacked in the stem, and the entire region from G7 to CIS in the upper stem and loop appears to be flexible. Several of these residues have a large fraction of S-puckered ribose rings, rather than the N-forms characteristic of RNA duplexes. There is considerable variation in the low-energy loop conformations that satisfy the distance constraints at this preliminary level of refinement. The Shine-Dalgarno ribosome binding site is exposed, and only two apparently weak base pairs would have to break for the 16s ribosomal RNA to bind and the ribosome to initiate translation of the replicase gene. Although the loop form must be regarded as tentative, the known interaction sites with the coat protein are easily accessible from the major groove side of the loop.
Ribose 2′-Hydroxyl Groups Stabilize RNA Hairpin Structures Containing GCUAA Pentaloop
Journal of Chemical Theory and Computation, 2013
The chemical structure of RNA and DNA is very similar; however, the three-dimensional conformation of these two nucleic acids is very different. Whereas the DNA adopts a repetitive structure of a double-stranded helix, RNA is primarily single stranded with a complex three-dimensional structure in which the hairpin is the most common secondary structure. Apart from the difference between uracil and thymine, the difference in the chemical structure between RNA and DNA is the presence of a hydroxyl group at position 2′ of the sugar (ribose) instead of a hydrogen (deoxyribose). In this paper, we present molecular dynamics simulations addressing the contribution of 2′-hydroxyls to the stability of a GCUAA pentaloop motif. The results indicate that the 2′-hydroxyls stabilize the hairpin conformation of the GCUAA pentaloop relative to an analogous oligonucleotide in which the ribose sugars in the loop region were substituted with deoxyriboses. The magnitude of the stabilization was found to be 23.8 ± 4.1 kJ/mol using an alchemical mutations free energy method and 4.2 ± 6.5 kJ/mol using potential of mean force calculations. The latter indicates that in addition to its larger thermodynamic stability the RNA hairpin is also kinetically more stable. We find that the excess stability is a result of intrahairpin hydrogen bonds in the loop region between the 2′-hydroxyls and sugars, bases, and phosphates. The hydrogen bonds with the sugars and phosphates involve predominantly interactions with adjacent nucleotides. However, the hydrogen bonds with the bases involve also interactions between groups on opposite sides of the loop or with the middle base of the loop and are therefore likely to contribute significantly to the stability of the loop. Of these hydrogen bonds, the most frequent is observed between the 2′-hydroxyl at the first position of the pentaloop with N6/N7 of adenine at the forth position, as well as between the 2′-hydroxyl at position −1 with N6 of adenine at the fifth position. Our results contribute to the notion that one of the important roles of the ribose sugars in RNA is to facilitate hairpin formation.
Biochemistry, 2017
The prediction of RNA three-dimensional structure from sequence alone has been a long-standing goal. High-resolution, experimentally determined structures of simple noncanonical pairings and motifs are critical to the development of prediction programs. Here, we present the nuclear magnetic resonance structure of the (5'CCAGAAACGGAUGGA)2 duplex, which contains an 8 × 8 nucleotide internal loop flanked by three Watson-Crick pairs on each side. The loop is comprised of a central 5'AC/3'CA nearest neighbor flanked by two 3RRs motifs, a known stable motif consisting of three consecutive sheared GA pairs. Hydrogen bonding patterns between base pairs in the loop, the all-atom root-mean-square deviation for the loop, and the deformation index were used to compare the structure to automated predictions by MC-sym, RNA FARFAR, and RNAComposer.
Structure of an RNA Internal Loop Consisting of Tandem C-A+ Base Pairs
Biochemistry, 1998
The crystal structure of the RNA octamer 5′-CGC(CA)GCG-3′ has been determined from X-ray diffraction data to 2.3 Å resolution. In the crystal, this oligomer forms a self-complementary double helix in the asymmetric unit. Tandem non-Watson-Crick C-A and A-C base pairs comprise an internal loop in the middle of the duplex, which is incorporated with little distortion of the A-form double helix. From the geometry of the C-A base pairs, it is inferred that the adenosine imino group is protonated and donates a hydrogen bond to the carbonyl group of the cytosine. The wobble geometry of the C-A + base pairs is very similar to that of the common U-G non-Watson-Crick pair. The picture of RNA molecules being structurally and functionally limited by the use of only four building blocks and by the rules of Watson-Crick base pairing is undergoing a radical change. Noncanonical base pairing within doublehelical regions of RNA has been structurally characterized (1-5) and shown to be functionally important as binding sites for protein (6, 7) and RNA (8), in active sites of catalytic RNA (9), and in bending RNA into a folded form (10, 11). Several of the noncanonical base pairs are polymorphic (12, 13), assuming a different pairing geometry depending on their environments or the solution conditions. We must also consider that at least some of the bases involved in noncanonical interactions may exist in different charged or protonated forms in different environments. For example, protonation of adenine and cytosine has been observed in the formation of C-A + (14) and CC + (15, 16) base pairs. We report here the 2.3 Å resolution (Table 1) X-ray structure of an RNA octamer which in the crystal forms a double helix incorporating tandem C-A-A-C base pairs bounded by standard Watson-Crick pairs. From the geometry of these noncanonical base pairs, it is inferred that they are C-A + pairs with a protonated N1 of the adenine donating a hydrogen bond to the cytosine carbonyl group. This "wobble" C-A + base pairing is structurally similar to, and in some cases may be a substitute for, the more common U-G wobble pair. MATERIALS AND METHODS Synthesis, Crystallization, and Data Collection. The octaribonucleotide rCGCCAGCG was chemically synthesized (Oligos, Etc.) and purified by anion exchange (DEAE-5PW TSK-Gel, TosoHaas) FPLC 1 with a linear salt gradient from 0.4 to 2.0 M sodium acetate and 50 mM Tris-HCl (pH 6.8-7.3). Crystals were grown at room temperature by the hanging drop vapor diffusion method from a 2.4 µL drop of a solution of 2 mM RNA mixed with an equal volume of the reservoir solution consisting of 20 mM NaCl, 5 mM MgCl 2 , 2 mM spermine tetrahydrochloride, and 35% MPD (2-methyl-2,4-pentanediol). The crystals were originally grown from a buffer of 50 mM sodium cacodylate (pH 6.5), but superior crystals were later grown from a buffer of 100 mM sodium cacodylate (pH 5.5). The crystals grown at pH 6.5 were thin hexagonal plates with a maximum dimension 100 µm, while those grown at the lower pH were rods that grew to dimensions of 350 µm × 40 µm × 40 µm. X-ray data were collected on a Rigaku R-axis IIC imaging plate system using CuKR radiation (λ) 1.5418 Å) and φ
Nucleic acids research, 2003
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 ...
Crystal Structure of a Conserved Ribosomal Protein-RNA Complex
Science, 1999
The structure of a highly conserved complex between a 58-nucleotide domain of large subunit ribosomal RNA and the RNA-binding domain of ribosomal protein L11 has been solved at 2.8 angstrom resolution. It reveals a precisely folded RNA structure that is stabilized by extensive tertiary contacts and contains an unusually large core of stacked bases. A bulge loop base from one hairpin of the RNA is intercalated into the distorted major groove of another helix; the protein locks this tertiary interaction into place by binding to the intercalated base from the minor groove side. This direct interaction with a key ribosomal RNA tertiary interaction suggests that part of the role of L11 is to stabilize an unusual RNA fold within the ribosome.