Imino proton NMR assignments and ion-binding studies on Escherichia coli tRNA3Gly

The imino region of the proton NMR spectrum of Escherichia coli tRNA3Gly has been assigned mainly by sequential nuclear Overhauser effects between neighbouring base pairs and by comparison of assignments of other tRNAs. The effects of magnesium, spermine and temperature on the 1H and 31P NMR spectra of this tRNA were studied. Both ions affect resonances close to the G15 . C48 tertiary base pair and in the ribosylthymine loop. The magnesium studies indicate the presence of an altered tRNA conformer at low magnesium concentrations in equilibrium with the high magnesium form. The temperature studies show that the A7 . U66 imino proton (from a secondary base pair) melts before some of the tertiary hydrogen bonds and that the anticodon stem does not melt sequentially from the ends. Correlation of the ion effects in the 1H and 31P NMR spectra has led to the tentative assignment of two 31P resonances not assigned in the comparable 31P NMR spectrum of yeast tRNAPhe. 31P NMR spectra of E. coli tRNA3Gly lack resolved peaks corresponding to peaks C and F in the spectra of E. coli tRNAPhe and yeast tRNAPhe. In the latter tRNAs these peaks have been assigned to phosphate groups in the anticodon loop. Ion binding E. coli tRNA3Gly and E. coli tRNAPhe had different effects on their 1H NMR spectra which may reflect further differences in their charge distribution and conformation.     

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.     

Solution conformation of an RNA hairpin loop.

The hairpin conformation adopted by the RNA sequence 5'GCGAUUUCUGACCGCC3' has been studied by one- and two-dimensional NMR spectroscopy. Exchangeable imino spectra in 60 mM Na+ indicate that the hairpin has a stem of six base pairs (indicated by boldface type) and a loop of three nucleotides. NOESY spectra of nonexchangeable protons confirm the formation of the stem region. The duplex has an A-conformation and contains an A.C apposition; a G.U base pair closes the loop region. The stem nucleotides have C3'-endo sugar conformations, as expected of an A-form duplex, whereas the three loop nucleotides adopt C2'-endo sugar puckers. Stacking within the loop, C8 upon the sugar of U7, stabilizes the structure. The pH dependence of both the exchangeable and nonexchangeable NMR spectra is consistent with the formation of an A+.C base pair, protonated at the N1 position of adenine. The stability of the hairpin was probed by using absorbance melting curves. The hairpin structure with the A+.C base pair is about +2 kcal/mol less stable in free energy at 37 degrees C than the hairpin formed with an A.U pair replacing the A+.C pair.     

Comparative analysis of hairpin ribozyme structures and interference data

Great strides in understanding the molecular underpinnings of RNA catalysis have been achieved with advances in RNA structure determination by NMR spectroscopy and X-ray crystallography. Despite these successes the functional relevance of a given structure can only be assessed upon comparison with biochemical studies performed on functioning RNA molecules. The hairpin ribozyme presents an excellent case study for such a comparison. The active site is comprised of two stems each with an internal loop that forms a series of non-canonical base pairs. These loops dock into each other to create an active site for catalysis. Recently, three independent structures have been determined for this catalytic RNA, including two NMR structures of the isolated loop A and loop B stems and a high-resolution crystal structure of both loops in a docked conformation. These structures differ significantly both in their tertiary fold and the nature of the non-canonical base pairs formed within each loop. Several of the chemical groups required to achieve a functioning hairpin ribozyme have been determined by nucleotide analog interference mapping (NAIM). Here we compare the three hairpin structures with previously published NAIM data to assess the convergence between the structural and functional data. While there is significant disparity between the interference data and the individual NMR loop structures, there is almost complete congruity with the X-ray structure. The only significant differences cluster around an occluded pocket adjacent to the scissile phosphate. These local differences may suggest a role for these atoms in the transition state, either directly in chemistry or via a local structural rearrangement.     

Solution structure of an ATP-binding RNA aptamer reveals a novel fold.

In vitro selection has been used to isolate several RNA aptamers that bind specifically to biological cofactors. A well-characterized example in the ATP-binding RNA aptamer family, which contains a conserved 11-base loop opposite a bulged G and flanked by regions of double-stranded RNA. The nucleotides in the consensus sequence provide a binding pocket for ATP (or AMP), which binds with a Kd in the micromolar range. Here we present the three-dimensional solution structure of a 36-nucleotide ATP-binding RNA aptamer complexed with AMP, determined from NMR-derived distance and dihedral angle restraints. The conserved loop and bulged G form a novel compact, folded structure around the AMP. The backbone tracing of the loop nucleotides can be described by a Greek zeta (zeta). Consecutive loop nucleotides G, A, A form a U-turn at the bottom of the zeta, and interact with the AMP to form a structure similar to a GNRA tetraloop, with AMP standing in for the final A. Two asymmetric G. G base pairs close the stems flanking the internal loop. Mutated aptamers support the existence of the tertiary interactions within the consensus nucleotides and with the AMP found in the calculated structures.     

A base-triple structural domain in RNA.

An oligonucleotide modeled on a proposed base-triple domain of the Tetrahymena group I intron has been characterized by NMR. The oligonucleotide contains two double-helix regions with adjacent single-stranded nucleotides. The NMR data show that the two helices stack coaxially, although the rotation between the two helices is approximately twice as large as the rotation between normal base pairs. The rotation between the two helices allows the single-stranded nucleotides to form U.U.G and A.G.C base triples in the minor groove. The A.G.C base triple contains a hydrogen bond between the adenine N1 and a 2'-hydroxyl in the minor groove of the G.C pair. A similar hydrogen bond between an adenine and a 2'-hydroxyl in transfer RNA suggests that this could be a recurring tertiary interaction in RNA.     

The structure of the L3 loop from the hepatitis delta virus ribozyme: a syn cytidine.

The structure of the L3 central hairpin loop isolated from the antigenomic sequence of the hepatitis delta virus ribozyme with the P2 and P3 stems from the ribozyme stacked on top of the loop has been determined by NMR spectroscopy. The 26 nt stem-loop structure contains nine base pairs and a 7 nt loop (5'-UCCUCGC-3'). This hairpin loop is critical for efficient catalysis in the intact ribozyme. The structure was determined using homonuclear and heteronuclear NMR techniques on non-labeled and15N-labeled RNA oligonucleotides. The overall root mean square deviation for the structure was 1.15 A (+/- 0.28 A) for the loop and the closing C.G base pair and 0.90 A (+/- 0.18 A) for the loop and the closing C.G base pair but without the lone purine in the loop, which is not well defined in the structure. The structure indicates a U.C base pair between the nucleotides on the 5'- and 3'-ends of the loop. This base pair is formed with a single hydrogen bond involving the cytosine exocyclic amino proton and the carbonyl O4 of the uracil. The most unexpected finding in the loop is a syn cytidine. While not unprecedented, syn pyrimidines are highly unusual. This one can be confidently established by intranucleotide distances between the ribose and the base determined by NMR spectroscopy. A similar study of the structure of this loop showed a somewhat different three-dimensional structure. A discussion of differences in the two structures, as well as possible sites of interaction with the cleavage site, will be presented.     

Factors affecting the thermodynamic stability of small asymmetric internal loops in RNA

Optical melting experiments were used to determine the thermodynamic parameters for oligoribonucleotides containing small asymmetric internal loops. The results show a broad range of thermodynamic stabilities, which depend on loop size, asymmetry, sequence, closing base pairs, and length of helix stems. Imino proton NMR experiments provide evidence for possible hydrogen bonding in GA and UU mismatches in some asymmetric loops. The stabilizing effects of GA, GG, and UU mismatches on the thermodynamic stability of internal loops vary depending on the size and asymmetry of the loop. The dependence of loop stability on Watson-Crick closing base pairs may be explained by an account of hydrogen bonds. Models are presented for approximating the free energy increments of 2 x 3 and 1 x 3 internal loops.     

The 5′ Stem-Loop From Human U4 snRNA Adopts a Novel Conformation Stabilized by G-C and G-U Base Pairs

Abstract

¹H NMR and molecular modeling studies of the 5′ stem-loop from human U4 snRNA were undertaken to determine the conformation of this stem-loop that is essential for spliceosome formation and pre-mRNA splicing. Sixteen of the 35 nucleotides of this stem-loop are in the loop region and inspection of the loop sequence revealed no decomposition into elements of secondary structure commonly found in other RNA stem-loops. An analysis of possible base pairing interactions for this stem-loop using the methods of Zuker revealed the lowest energy secondary structure for the 16 nucleotide loop consisted of four base pairs at the base of a non-canonical tetraloop (UUUA). This shorter stem-loop was joined to the nine base pair stem by two A residues on the 5′ side and a single bulged A on the 3′ side. Both stems also had bulged A residues. ¹H NMR experiments performed on solutions of the 35mer stem-loop, the stem region, and the loop region confirmed the 35mer adopted this secondary structure in solution. A 3D molecular model of this structure consistent with the NMR data was generated to assist in visualization of this novel structure.     

Structure of protein L7Ae bound to a K-turn derived from an archaeal box H/ACA sRNA at 1.8 A resolution

The archaeal RNA binding protein L7Ae and its eukaryotic homolog 15.5 kDa/Snu13 recognize K-turns. This structural motif is canonically comprised of two stems (one with tandem A.G base pairs, the other with Watson-Crick pairs) linked by an asymmetric internal loop. L7Ae recognizes conventional K-turns in ribosomal and box C/D RNAs but also binds specifically to some box H/ACA RNAs at terminal stem loops. These have the A.G paired stem, but lack the Watson-Crick stem. The structure of Methanococcus jannaschii L7Ae bound to a symmetric duplex RNA without Watson-Crick stems demonstrates how a binding site for this component of diverse ribonucleoprotein complexes can be constructed with only the A.G stem and the loop. The RNA adopts a functional conformation with the aid of a base triple and tight binding of divalent cations. Comparison with the 15.5 kDa/Snu13-RNA complex structure suggests why the eukaryotic homolog does not recognize terminal stem loop L7Ae binding sites.     

Loss of G-A base pairs is insufficient for achieving a large opening of U4 snRNA K-turn motif

Upon binding to the 15.5K protein, two tandem-sheared G–A base pairs are formed in the internal loop of the kink-turn motif of U4 snRNA (Kt-U4). We have reported that the folding of Kt-U4 is assisted by protein binding. Unstable interactions that contribute to a large opening of the free RNA (‘k–e motion’) were identified using locally enhanced sampling molecular dynamics simulations, results that agree with experiments. A detailed analysis of the simulations reveals that the k–e motion in Kt-U4 is triggered both by loss of G–A base pairs in the internal loop and backbone flexibility in the stems. Essential dynamics show that the loss of G–A base pairs is correlated along the first mode but anti-correlated along the third mode with the k–e motion. Moreover, when enhanced sampling was confined to the internal loop, the RNA adopted an alternative conformation characterized by a sharper kink, opening of G–A base pairs and modified stacking interactions. Thus, loss of G–A base pairs is insufficient for achieving a large opening of the free RNA. These findings, supported by previously published RNA structure probing experiments, suggest that G–A base pair formation occurs upon protein binding, thereby stabilizing a selective orientation of the stems.     

NMR studies on DNA hairpin structures with a five nucleotide loop

One- and two-dimensional NMR experiments have been undertaken to investigate the structure of DNA hairpins with a five nucleotide loop. Analysis of proton NMR spectra suggests that the four hairpin structures examined have some common structural features; B-type conformation in the stem region and the same stacking pattern, 5' (XXX-turn-XX) 3', in the loop region. The phosphorus NMR spectra suggest that the conformational changes in the loop region affect the backbone conformation of the stem duplex.     

An antibiotic-binding motif of an RNA fragment derived from the A-site-related region of Escherichia coli 16S rRNA.

A small RNA derived from the decoding region of Escherichia coli 16S rRNA can bind to antibiotics of aminoglycosides (neomycin and paromomycin) that act on the small ribosomal subunit [Purohit,P. and Stern,S. (1994) Nature, 370, 659-662]. In the present study, the P-site subdomain was removed from this decoding region RNA to construct a 27mer RNA (designated as ASR-27), which includes the A-site-related region (positions 1402-1412 and 1488-1497) of 16S rRNA. Footprint experiments with dimethyl sulfate as a chemical probe indicated that the ASR-27 RNA can interact with the neomycin family in the same manner as the decoding region RNA. A mutagenesis analysis of the ASR-27 RNA revealed that paromomycin binding of ASR-27 involves the C1407.G1494 and C1409-G1491 base pairs, and the internal loop comprising A1408 and the nucleotides in positions 1492-1493, located between the two C.G base pairs. In addition, a G or U in position 1495, and base pairing between positions 1405 and 1496 are also involved. These structural features were found in a viral RNA element, the Rev-binding site of human immunodeficiency virus type-1, which may explain why neomycin can bind to this viral RNA.     

Morpholino spin-labeling for base-pair sequencing of a 3'-terminal RNA stem by proton homonuclear Overhauser enhancements: yeast ribosomal 5S RNA

Base-pair sequences for 5S and 5.8S RNAs are not readily extracted from proton homonuclear nuclear Overhauser enhancement (NOE) connectivity experiments alone, due to extensive peak overlap in the downfield (11-15 ppm) proton NMR spectrum. In this paper, we introduce a new method for base-pair proton peak assignment for ribosomal RNAs, based upon the distance-dependent broadening of the resonances of base-pair protons spatially proximal to a paramagnetic group. Introduction of a nitroxide spin-label covalently attached to the 3'-terminal ribose provides an unequivocal starting point for base-pair hydrogen-bond proton NMR assignment. Subsequent NOE connectivities then establish the base-pair sequence for the terminal stem of a 5S RNA. Periodate oxidation of yeast 5S RNA, followed by reaction with 4-amino-2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO-NH2) and sodium borohydride reduction, produces yeast 5S RNA specifically labeled with a paramagnetic nitroxide group at the 3'-terminal ribose. Comparison of the 500-MHz 1H NMR spectra of native and 3'-terminal spin-labeled yeast 5S RNA serves to identify the terminal base pair (G1 . C120) and its adjacent base pair (G2 . U119) on the basis of their proximity to the 3'-terminal spin-label. From that starting point, we have then identified (G . C, A . U, or G . U) and sequenced eight of the nine base pairs in the terminal helix via primary and secondary NOE's.     

An NMR study of the helix V-loop E region of the 5S RNA from Escherichia coli

Experiments are described that complete the assignment of the imino proton NMR spectrum of the fragment 1 domain from the 5S RNA of Escherichia coli. Most of the new assignments fall in the helix V-loop E portion of the molecule (bases 70-78 and 98-106), the region most sensitive to the binding of ribosomal protein L25. The spectroscopic data are incompatible with the standard, phylogenetically derived model for 5S RNA, which makes all the base pairs possible in loop E with the sequences aligned in parallel (C70-G106, C71-G105, etc.) [see Delihas et al. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31, 161-190]. Furthermore, the alternative loop E model proposed for spinach chloroplast 5S RNA by Romby et al. [(1988) Biochemistry 27, 4721-4730] does not apply to the closely homologous 5S RNA from E. coli. The 5S RNAs from E. coli and spinach chloroplasts do not have the same secondary structures in solution despite their strong sequence homologies, and neither appears to conform to the standard model for 5S RNA in the loop E region.     

500-MHz proton homonuclear Overhauser evidence for additional base pair in the common arm of eukaryotic ribosomal 5S RNA: wheat germ

A "common-arm" fragment from wheat germ (Triticum aestivum) 5S RNA has been produced by enzymatic cleavage with RNase T1 and sequenced via autoradiography of electrophoresis gels for the end-labeled fragments obtained by further RNase T1 partial digestion. The existence, base pair composition, and base pair sequence of the common arm are demonstrated for the first time by means of proton 500-MHz nuclear magnetic resonance. From Mg2+ titration, temperature variation, ring current calculations, sequence comparisons, and proton homonuclear Overhauser enhancement experiments, additional base pairs in the common arm of the eukaryotic 5S RNA secondary structure are detected. Two base pairs, G41 X C34 and A42 X U33 in the hairpin loop, could account for the lack of binding between the conserved GAAC segment of 5S RNA and the conserved Watson-Crick-complementary GT psi C segment of tRNAs.     

Identification of three G.U base pairs in Bacillus subtilis ribosomal 5S RNA via 500-MHz proton homonuclear Overhauser enhancements

Three distinct G.U base pairs in Bacillus subtilis 5S RNA have been identified via homonuclear Overhauser enhancements (NOE) of their low-field (9-15 ppm) proton Fourier transform nuclear magnetic resonances at 11.75 T. With these G.U resonances as starting points, short segments of NOE connectivity can be established. One G.U-G.C-G.C segment (most probably G4.C112-G5.C111-U6.G110) can definitely be assigned to the terminal helix. The existence of at least part of the terminal helical stem of the secondary structure of a Gram-positive bacterial 5S RNA has thus been established for the first time by direct experimental observation. Addition of Mg2+ produces almost no conformational changes in the terminal stem but results in major conformational changes elsewhere in the structure, as reflected by changes in the 1H 500-MHz low-field NMR spectrum. Assignment of the two remaining G.U base pairs will require further experiments (e.g., enzymatic-cleavage fragments). Finally, the implications of these results for analysis of RNA secondary structure are discussed.     

RNA Sequence Determinants for Aminoglycoside Binding to an A-site rRNA Model Oligonucleotide

The codon-anticodon interaction on the ribosome occurs in the A site of the 30 S subunit. Aminoglycoside antibiotics, which bind to ribosomal RNA in the A site, cause misreading of the genetic code and inhibit translocation. Biochemical studies and nuclear magnetic resonance spectroscopy were used to characterize the interaction between the aminoglycoside antibiotic paromomycin and a small model oligonucle otide that mimics the A site ofEscherichia coli16 S ribosomal RNA. Upon chemical modification, the RNA oligonucleotide exhibits an accessibility pattern similar to that of 16 S rRNA in the 30 S subunit. In addition, the oligonucleotide binds specifically aminoglycoside antibiotics. The anti biotic binding site forms an asymmetric internal loop, caused by non-canonical base-pairs. Nucleotides that are important for binding of paromomycin were identified by performing quantitative footprinting on oligonucleotide sequence variants and include the C1407·G1494 base-pair, and A·U base-pair at positions 1410/1490, and nucleotides A1408, A1493 and U1495. The asymmetry of the internal loop, which requires the presence of a nucleotide in position 1492, is also crucial for antibiotic binding. Introduction into the oligonucleotide of base changes that are known to confer aminoglycoside resistance in 16 S rRNA result in weaker binding of paromomycin to the oligonucleotide. Oligonucleotides homologous to eukaryotic rRNA sequences show reduced binding of paromomycin, suggesting a physical origin for the species-specific action of aminoglycosides.     

Evaluation of base-pairing schemes for E. coli 5S RNA by 400 MHz proton nuclear magnetic resonance spectroscopy

The number of base pairs in the denatured “B” form of $\text{E. coli}$ 5S RNA has been determined directly from 400 MHz high resolution proton nuclear magnetic resonance spectroscopy. The experimental NMR spectrum from ?11.6 to ?14.5 ppm from a sodium 2,2-dimethyl-2-silapentane sulfonate reference can be simulated by a theoretical spectrum consisting of 33 Lorentzian lines of equal width (corresponding to 33 base pairs) at 26°C. This result is inconsistent with previously proposed secondary structures of 17 and 23 base pairs, but is readily adapted to the Luoma-Marshall cloverleaf secondary structure.