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.     

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.     

Demonstration of the GC-rich common arm in yeast ribosomal 5.8S RNA via 500-MHz proton nuclear magnetic resonance and Overhauser enhancements

In this paper we report the first 1H NMR study of the base-paired secondary structure of yeast 5.8S RNA. On the basis of a combination of homonuclear Overhauser enhancements and temperature dependence of the proton 500-MHz NMR spectrum, we are able to identify and assign eight of the nine base pairs in the most thermally stable helical arm: G116.C137-C117.G136-C118.G135- C119.G134-C120.G133-U121.G132- U122.A131-G123.C130. This arm contains an unusually temperature-stable (to 71 degrees C) segment of four consecutive G.C base pairs. This work constitutes the most direct evidence to date for the existence and base-pair sequence of the GC-rich helix, which is common to most currently popular secondary structural models for eukaryotic 5.8S ribosomal RNA.     

Identification and assignment of base pairs in the "tuned helix" of intact and ribonuclease T1 cleavage fragments of wheat germ ribosomal 5S RNA via 500-MHz proton homonuclear Overhauser enhancements

Wheat germ has been chosen as a representative eukaryote for study of ribosomal 5S RNA secondary structure. Proton homonuclear Overhauser enhancements (NOE's) at 500 MHz for the hydrogen-bonded base-pair protons in the 10-15 ppm region are used to establish the identity (A X U, G X C, or G X U) and base-pair sequence (e.g., G X C-A X U-C X G) within a given helical segment. Assignment of that segment to particular base pairs in the secondary structure is based upon NOE's conducted at different temperatures (to determine which signals "melt" together), variation of salt conditions (to produce differential chemical shifts in order to better distinguish components of an unresolved spectral envelope), and isolation and purification of RNase T1 cleavage fragments (in order to reduce the spectrum to just a few base pairs). The NOE patterns for the RNase T1 fragments are the same as in the intact 5S RNA, supporting the assumption that structural features of this region in the intact 5S RNA are preserved in the fragment. Chemical shifts predicted from ring current induced effects for a proposed base-pair sequence are then compared to experimental chemical shifts. By these methods, a portion of the "tuned helix" segment (namely, the base-pair sequence C18G60-A19U59-C20G58) is demonstrated spectroscopically for the first time in any 5S RNA. The tuned helix and common arm segments are less stable than the rest of the molecule. Variation of sodium and magnesium levels reveals multiple configurations of the wheat germ 5S RNA in solution.     

Identification and assignment of base pairs in four helical segments of Bacillus megaterium ribosomal 5S RNA and its ribonuclease T1 cleavage fragments by means of 500-MHz proton homonuclear Overhauser enhancements.

Three different fragments of Bacillus megaterium ribosomal 5S RNA have been produced by enzymatic cleavage with ribonuclease T1. Fragment A consists of helices II and III, fragment B contains helix IV, and fragment C contains helix I of the universal 5S rRNA secondary structure. All (eight) imino proton resonances in the downfield region (9-15 ppm) of the 500-MHz proton FT NMR spectrum of fragment B have been identified and assigned as G80.C92-G81.C91-G82.C90-A83.++ +U89-C84.G88 and three unpaired U's (U85, U86, and U87) in helix IV by proton homonuclear Overhauser enhancement connectivities. The secondary structure in helix IV of the prokaryotic loop is completely demonstrated spectroscopically for the first time in any native or enzyme-cleaved 5S rRNA. In addition, G21.C58-A20.U59-G19.C60-A18.U61 in helix II, U32.A46-G31.C47-C30.G48-C29.G49 in helix III, and G4.C112-G5.C111-U6.G110 in the terminal stem (helix I) have been assigned by means of NOE experiments on intact 5S rRNA and its fragments A and C. Base pairs in helices I-IV of the universal secondary structure of B. megaterium 5S RNA are described.     

Structural characterization of a six-nucleotide RNA hairpin loop found in Escherichia coli, r(UUAAGU)

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: DeltaH degrees = -42.8 +/- 2.2 kcal/mol, DeltaS degrees = -127.6 +/- 6.5 eu, and DeltaG degrees (37) = -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. T., Brodersen, D. E., Clemons, W. M., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Nature 407, 327-339]. The similarity of the solution and solid-state structures of this hairpin loop suggests that formation of this hairpin may facilitate folding of 16S RNA.     

Genetic interactions among the West Nile virus methyltransferase, the RNA-dependent RNA polymerase, and the 5' stem-loop of genomic RNA

Flavivirus methyltransferase catalyzes both guanine N7 and ribose 2'-OH methylations of the viral RNA cap (GpppA-RNA-->m(7)GpppAm-RNA). The methyltransferase is physically linked to an RNA-dependent RNA polymerase (RdRp) in the flaviviral NS5 protein. Here, we report genetic interactions of West Nile virus (WNV) methyltransferase with the RdRp and the 5'-terminal stem-loop of viral genomic RNA. Genome-length RNAs, containing amino acid substitutions of D146 (a residue essential for both cap methylations) in the methyltransferase, were transfected into BHK-21 cells. Among the four mutant RNAs (D146L, D146P, D146R, and D146S), only D146S RNA generated viruses in transfected cells. Sequencing of the recovered viruses revealed that, besides the D146S change in the methyltransferase, two classes of compensatory mutations had reproducibly emerged. Class 1 mutations were located in the 5'-terminal stem-loop of the genomic RNA (a G35U substitution or U38 insertion). Class 2 mutations resided in NS5 (K61Q in methyltransferase and W751R in RdRp). Mutagenesis analysis, using a genome-length RNA and a replicon of WNV, demonstrated that the D146S substitution alone was lethal for viral replication; however, the compensatory mutations rescued replication, with the highest rescuing efficiency occurring when both classes of mutations were present. Biochemical analysis showed that a low level of N7 methylation of the D146S methyltransferase is essential for the recovery of adaptive viruses. The methyltransferase K61Q mutation facilitates viral replication through improved N7 methylation activity. The RdRp W751R mutation improves viral replication through an enhanced polymerase activity. Our results have clearly established genetic interactions among flaviviral methyltransferase, RdRp, and the 5' stem-loop of the genomic RNA.     

An RNA domain within the 5' untranslated region of the tomato bushy stunt virus genome modulates viral RNA replication

The terminal half of the 5' untranslated region (UTR) in the (+)-strand RNA genome of tomato bushy stunt virus was analyzed for possible roles in viral RNA replication. Computer-aided thermodynamic analysis of secondary structure, phylogenetic comparisons for base-pair covariation, and chemical and enzymatic solution structure probing were used to analyze the 78 nucleotide long 5'-terminal sequence. The results indicate that this sequence adopts a branched secondary structure containing a three-helix junction core. The T-shaped domain (TSD) formed by this terminal sequence is closed by a prominent ten base-pair long helix, termed stem 1 (S1). Deletion of either the 5' or 3' segment forming S1 (coordinates 1-10 or 69-78, respectively) in a model subviral RNA replicon, i.e. a prototypical defective interfering (DI) RNA, reduced in vivo accumulation levels of this molecule approximately 20-fold. Compensatory-type mutational analysis of S1 within this replicon revealed a strong correlation between formation of the predicted S1 structure and efficient DI RNA accumulation. RNA decay studies in vivo did not reveal any notable changes in the physical stabilities of DI RNAs containing disrupted S1s, thus implicating RNA replication as the affected process. Further investigation revealed that destabilization of S1 in the (+)-strand was significantly more detrimental to DI RNA accumulation than (-)-strand destabilization, therefore S1-mediated activity likely functions primarily via the (+)-strand. The essential role of S1 in DI RNA accumulation prompted us to examine the 5'-proximal secondary structure of a previously identified mutant DI RNA, RNA B, that lacks the 5' UTR but is still capable of low levels of replication. Mutational analysis of a predicted S1-like element present within a cryptic 5'-terminal TSD confirmed the importance of the former in RNA B accumulation. Collectively, these data support a fundamental role for the TSD, and in particular its S1 subelement, in tombusvirus RNA replication.     

Terminal nucleotide sequences of 17-S ribosomal RNA and its immediate precursor 18-S RNA in yeast.

The 5' and 3'-terminal nucleotide sequences of 17-S rRNA and its immediate precursor 18-S RNA from the yeast Saccharomyces carlsbergensis have been analysed. Identification of the terminal oligonucleotides, as present in Ti ribonuclease digests, was performed by diagonal procedures. The major (molar yield 0.9) 5'-terminal oligonucleotide (molar yield 0.15) with the overall composition pU (U2,C2)G was observed. 18-S precursor RNA was found to contain the same 5'-terminal sequences as 17-S rRNA. However, the 3'-terminal sequences of the two types of RNA appeared to be different. The 17-S rRNA yields the oligonucleotide A-U-C-A-U-U-AOH while at least half of the 18-S RNA molecules contain the sequence U-U-U-C-A-A-U-AOH. In addition 18-S RNA yields several minor 3'-terminal oligonucleotides which appear to be structurally related to the major 3'-terminal sequence. These results demonstrate that the extra nucleotides in 18-S RNA relative to 17-S RNA are located exclusively at the 3'-terminus of the 18-S RNA molecule. The possibility that the 3'-terminal nucleotide sequence of 18-S RNA plays a role in the maturation process is discussed.     

Flexibility of end-labeled polymers from electron spin resonance line-shape analysis: 3' terminus of transfer ribonucleic acid and 5S ribonucleic acid

Saccharomyces cerevisiae tRNA and 5S RNA, Escherichia coli 5S RNA, and wheat germ 5S RNA have each been specifically spin-labeled at the 3'-terminal ribose to give morpholino-spin-labeled (MSL) RNAs. Enzymatic hydrolysis with pancreatic RNase, followed by anion-exchange chromatography, confirms the site of attachment of the spin-label. Effective rotational correlation times, TB and TC, have been determined from electron spin resonance (ESR) peak heights and widths as a function of temperature for each MSL RNA, and Arrhenius plots of -log T vs. 1/T have been constructed. TC is a measure of internal flexibility at the link between the label and the RNA, while TB is a measure of rotational flexibility of the RNA near the labeled site. Validity of the TB and TC determination has been confirmed from simulation of the experimental EPR spectra by theoretical spectra computed for various attachment geometries and motional rates. Discontinuities in the slope of Arrhenius plots for TB were seen at 34 and 66 degrees C (yeast MSL tRNA), 37 and 60 degrees C (E. coli MSL 5S RNA), 37 and 57 degrees C (yeast MSL 5S RNA), and 36 and 54 degrees C (wheat germ MSL 5S RNA). Temperature-induced hydrolysis of each MSL RNA was less than 5% as determined by gel-filtration chromatography. The melting curves are consistent with a recently proposed universal secondary structural model for prokaryotic and eukaryotic 5S RNA.     

Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution

In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5′-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA.     

Conformational and thermodynamic effects of naturally occurring base methylations in a ribosomal RNA hairpin of Bacillus stearothermophilus.

The 3'-terminal colicin fragments of 16S ribosomal RNA were isolated from Bacillus stearothermophilus and from its kasugamycin-resistant (ksgA) derivative lacking N6-dimethylation of the two adjacent adenosines in a hairpin loop. The fragment from the ksgA strain still contains a naturally occurring N2-methylguanosine in the loop. An RNA molecule resembling the B. stearothermophilus colicin fragment but without modified nucleosides was synthesized in vitro using a DNA template and bacteriophage T7 RNA polymerase. Proton-NMR spectra of the RNAs were recorded at 500 MHz. The imino-proton resonances of base-paired G and U residues could be assigned on the basis of previous NMR studies of the colicin fragment of Escherichia coli and by a combination of methylation-induced shifts and thermal melting of base pairs. The assignments were partly confirmed by NOE measurements. Adenosine dimethylation in the loop has a distinct conformational effect on the base pairs adjoining the loop. The thermal denaturation melting curve of the enzymatically synthesized RNA fragment was also determined and the transition midpoint (tm) was found to be 73 degrees C at 15 mM Na+. A comparison with previously determined thermodynamic parameters for various colicin fragments demonstrates that base methylations in the loop lead to a relatively strong destabilization of the hairpin helix. In terms of free energy the positive contribution of the methylations are in the order of the deletion of one base pair from the stem. Other data show that recently published free-energy parameters do not apply for certain RNA hairpins.     

Conformation and dynamics of an RNA internal loop

The conformation and the dynamics of an RNA oligonucleotide (26 nucleotides) which is a model for loop E in eukaryotic 5S RNA have been investigated by one- and two-dimensional NMR. The central portion of the oligonucleotide contains two G A oppositions, a common feature of ribosomal RNAs. The exchangeable proton spectrum indicates that an internal loop separates two stems of four and five base pairs. This observation is not consistent with structures for loop E containing mismatched G.A base pairs proposed from chemical and enzymatic studies on Xenopus laevis 5S RNA. The nonexchangeable proton spectrum has been assigned by two-dimensional NMR. Scalar couplings from correlated experiments and interproton distances from NOESY experiments at short mixing times have been used to determine glycosidic angles, sugar puckers, and other conformational features. The conformation of the stems is very close to standard A-form RNA, and extensive base stacking continues into the internal loop. This result provides a structural basis for the large favorable enthalpy of duplex formation determined in thermodynamic studies. Unusual structural and dynamic features are localized in the nucleotides connecting the loop to the stems.     

Resolution enhanced homonuclear carbon decoupled triple resonance experiments for unambiguous RNA structural characterization

Large RNAs (>30 nucleotides) suffer from extensive resonance overlap that can seriously hamper unambiguous structural characterization. Here we present a set of 3D multinuclear NMR experiments with improved and optimized resolution and sensitivity for aiding with the assignment of RNA molecules. In all these experiments strong base and ribose carbon–carbon couplings are eliminated by homonuclear band-selective decoupling, leading to improved signal to noise and resolution of the C5, C6, and C1′ carbon resonances. This decoupling scheme is applied to base-type selective ¹³C-edited NOESY, ¹³C-edited TOCSY (HCCH, CCH), HCCNH, and ribose H1C1C2 experiments. The 3D implementation of the HCCNH experiment with both carbon and nitrogen evolution enables direct correlation of ¹³C and ¹⁵N resonances at different proton resonant frequencies. The advantages of the new experiments are demonstrated on a 36 nucleotides hairpin RNA from domain 5 (D5) of the group II intron Pylaiella littoralis using an abbreviated assignment strategy. These four experiments provided additional separation for regions of the RNA that have overlapped chemical shift resonances, and enabled the assignment of critical D5 bulge nucleotides that could not be assigned using current experimental schemes.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-5093-6     

Database proton NMR chemical shifts for RNA signal assignment and validation

The Biological Magnetic Resonance Data Bank contains NMR chemical shift depositions for 132 RNAs and RNA-containing complexes. We have analyzed the ¹H NMR chemical shifts reported for non-exchangeable protons of residues that reside within A-form helical regions of these RNAs. The analysis focused on the central base pair within a stretch of three adjacent base pairs (BP triplets), and included both Watson–Crick (WC; G:C, A:U) and G:U wobble pairs. Chemical shift values were included for all 4³ possible WC-BP triplets, as well as 137 additional triplets that contain one or more G:U wobbles. Sequence-dependent chemical shift correlations were identified, including correlations involving terminating base pairs within the triplets and canonical and non-canonical structures adjacent to the BP triplets (i.e. bulges, loops, WC and non-WC BPs), despite the fact that the NMR data were obtained under different conditions of pH, buffer, ionic strength, and temperature. A computer program (RNAShifts) was developed that enables convenient comparison of RNA ¹H NMR assignments with database predictions, which should facilitate future signal assignment/validation efforts and enable rapid identification of non-canonical RNA structures and RNA-ligand/protein interaction sites.     

Base pairing in wheat germ ribosomal 5S RNA as measured by ultraviolet absorption, circular dichroism, and Fourier-transform infrared spectrometry

Ultraviolet absorption (UV) and circular dichroism (CD) spectra of wheat germ 5S RNA, when compared to tRNAPhe, indicate a largely base-paired and base-stacked helical structure, containing up to 36 base pairs. Fourier-transform infrared (FT-IR) spectra of tRNAPhe and wheat germ ribosomal 5S RNA have been acquired at 30 and 90 degrees C. From the difference of the FT-IR spectra between 90 and 30 degrees C, the number of base pairs in both RNAs was determined by modification of a previously published procedure [Burkey, K. O., Marshall, A. G., & Alben, J. O. (1983) Biochemistry 22, 4223-4229]. The base-pair composition and total base-pair number from FT-IR data are now consistent for the first time with optical (UV, CD, Raman) and NMR results for ribosomal 5S RNA. Without added Mg2+, tRNAPhe gave 18 +/- 2 base pairs [7 A-U and 11 G-C], in good agreement with the number of secondary base pairs from X-ray crystallography [8 A-U, 12 G-C, and 1 G-U]. Within the 10% precision of the FT-IR method, wheat germ 5S RNA exhibits essentially the same number of base pairs [14 A-U, 17 G-C, and 5 G-U; for a total of 36] in the absence of Mg2+ as in the presence of Mg2+ [14 A-U, 18 G-C, and 3 G-U; for a total of 35], in agreement with the UV hyperchromism estimate of G-C/(A-U + G-C) = 0.58.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural studies on tRNA acceptor stem microhelices: exchange of the discriminator base A73 for G in human tRNALeu switches the acceptor specificity from leucine to serine possibly by decreasing the stability of the terminal G1-C72 base pair.

Correct recognition of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (aaRS) is crucial to the maintenance of translational fidelity. The discriminator base A73 in human tRNALeuis critical for its specific recognition by the aaRS. Exchanging A73 for G abolishes leucine acceptance and converts it into a serine acceptor in vitro . Two RNA microhelices of 24 nt length that correspond to the tRNALeuacceptor stem and differ only in the discriminator base were synthesized: a wild-type tRNALeumicrohelix, where nt 21 corresponds to the discriminator base position 73, and an A21G mutant microhelix. To investigate whether different identities of both tRNAs are caused by conformational differences, NMR and UV melting experiments were performed on both microhelices. Two-dimentional NOESY spectra showed both microhelices to exhibit the same overall conformation at their 3'-CCA ends. Thermodynamic analysis and melting behaviour of the base-paired imino protons observed by NMR spectroscopy suggest that the A21G (A73G in tRNA) exchange results in a decrease of melting transition cooperativity and a destabilization of the terminal G1-C20 (G1-C72 in tRNA) base pair. Furthermore, the fact that this 3'-terminal imino proton is more solvent-exposed at physiological temperature might be another indication for the importance of the stability of the terminal base pair for specific tRNA recognition.