On the base-stacking in the 5'-terminal cap structure of mRNA: a fluorescence study.

The fluorescence at 370 nm of the 7-methylguanosine residue (m7G) is found to be quenched when the base residue is involved in a stacking interaction with the adenosine residue in the cap structure m7G5' pppA of an eukaryotic mRNA. On the basis of the observed degree of quenching, the amounts of the stacked and unstacked forms in the cap structure have been determined at various temperatures and pH's. It has been found that at pH 6.2 effective enthalpy and entropy in the unstacked leads to stacked change are delta H degrees = 4.4 +/- 0.1 kcal/mole and delta S degrees = - 14.3 +/- 0.2 e.u., respectively. The pka value for the m7G residue is found to be 7.7 at 10 degrees C and 7.3 at 30 degrees C. The stacked structure seems to be less favourable in the deprotonated form that occurs in the higher pH solution. A similar analysis of some other cap structures indicates that the stacked form in m7G5' pppN structure is favourable if N is a purine nucleoside or a 2'-O-methylpyrimidine nucleoside but not for an unmethylated pyrimidine nucleoside.     

Stabilization of RNA stacking by pseudouridine.

The effect of the modified nucleoside pseudouridine (psi) on RNA structure was compared with uridine. The extent of base stacking in model RNA oligonucleotides was measured by 1H NMR, UV, and CD spectroscopy. The UV and CD results indicate that the model single-stranded oligoribonucleotides AAUA and AA psi A form stacked structures in solution and the CD results for AA psi A are consistent with a general A-form helical conformation. The AA psi A oligomer exhibits a greater degree of UV hypochromicity over the temperature range 5-55 degrees C, consistent with a better stacked, more A-form structure compared with AAUA. The extent of stacking for each nucleotide residue was inferred from the percent 3'-endo sugar conformation as indicated by the H1'-H2' NMR scalar coupling. This indirect indication of stacking was confirmed by sequential NOE experiments. NMR measurements as a function of temperature indicate that pseudouridine forms a more stable base stacking arrangement than uridine, an effect that is propagated throughout the helix to stabilize stacking of neighboring purine nucleosides. The N1-H imino proton in AA psi A exchanges slowly with solvent, suggesting a role for the extra imino proton in stabilizing the conformation of pseudouridine. These results show that the conformational stabilization is an intrinsic property of pseudouridine occurring at the nucleotide level. The characteristics of pseudouridine in these models are consistent with earlier studies on intact rRNA, indicating that pseudouridine probably performs the same stabilizing function in most structural contexts.     

Conformational analysis of oligoarabinonucleotides. An NMR and CD study.

A 500 and 300 MHz proton NMR study of the series of oligoarabinonucleotides 5'aAMP, 3'aAMP, aA-aA, (aA-)2aA and (aA-)3aA is presented. In addition, circular dichroism is used to study the stacking behaviour of aA-aA. The complete 1H-NMR spectral assignment of the compounds (except the tetramer) is given. Proton-proton and proton-phosphorus coupling constants, obtained by computer simulation of the high-field region of the spectra, yield information on the conformation of the arabinose rings (N- or S-type) and on the intramolecular stacking properties of the dimer and the trimer. The monomers 5'aAMP and 3'aAMP exhibit a preference for N- and S-type sugar conformation, respectively. It is shown that the dimer aA-aA at low temperature prefers a mixed stacked state of the type aA(S)-aA(N). In the trimer the aA(2)-aA(3) fragment exhibits a conformation similar to that found in the dimer, whereas the aA(1) residue prefers to adopt S-type sugar and has some tendency to stack upon residue aA(2).     

Spectroscopic and theoretical insights into sequence effects of aminofluorene-induced conformational heterogeneity and nucleotide excision repair

A systematic spectroscopic and computational study was conducted in order to probe the influence of base sequences on stacked (S) versus B-type (B) conformational heterogeneity induced by the major dG adduct derived from the model carcinogen 7-fluoro-2-aminofluorene (FAF). We prepared and characterized eight 12-mer DNA duplexes (-AG*N- series, d[CTTCTAG*NCCTC]; -CG*N- series, d[CTTCTCG*NCCTC]), in which the central guanines (G*) were site-specifically modified with FAF with varying flanking bases (N = G, A, C, T). S/B heterogeneity was examined by CD, UV, and dynamic 19F NMR spectroscopy. All the modified duplexes studied followed a typical dynamic exchange between the S and B conformers in a sequence dependent manner. Specifically, purine bases at the 3'-flanking site promoted the S conformation (G > A > C > T). Simulation analysis showed that the S/B energy barriers were in the 14-16 kcal/mol range. The correlation times (tau = 1/kappa) were found to be in the millisecond range at 20 degrees C. The van der Waals energy force field calculations indicated the importance of the stacking interaction between the carcinogen and neighboring base pairs. Quantum mechanics calculations showed the existence of correlations between the total interaction energies (including electrostatic and solvation effects) and the S/B population ratios. The S/B equilibrium seems to modulate the efficiency of Escherichia coli UvrABC-based nucleotide excision repair in a conformation-specific manner: i.e., greater repair susceptibility for the S over B conformation and for the -AG*N- over the -CG*N- series. The results indicate a novel structure-function relationship, which provides insights into how bulky DNA adducts are accommodated by UvrABC proteins.     

Structural and conformational studies on deoxyguanosyl-3',5'-deoxyadenosine monophosphate and its ethyl phosphotriester analogs--left-handed dimers

The mode of base-base stacking, the handedness and the sugar(dGpA)phosphate backbone conformation of deoxyguanosyl 3'-5' deoxyadenosine and its diastereomeric ethyl phosphotriester analogs were studied by 1H NMR, UV and CD spectroscopy. The results indicate the three dimers are left-handed, while the sugar phosphate backbone is comprised predominantly of C2-endo,gg(C4-C5) and g'g (C5-O) conformers. The two bases are extensively stacked and interact about 90 degrees along the dyad axes. The extent of base overlap in dGpA is slightly greater than in either ethyl phosphotriester analog. The absolute configurations of the two ethyl phosphotriester diastereoisomers of dGpA can be assigned by one-dimensional and two-dimensional 1H NMR nuclear Overhauser enhancement experiments.     

Synthesis and properties of ApU analogues containing 2'-halo-2'-deoxyadenosines. Effects of 2' substituents on oligonucleotide conformation

Five A-U analogues containing deoxyadenosine or 2'-halo-2'-deoxyadenosines, which are known to have widely different C3'-endo conformer populations according to their electronegativities of the halogen substituents, dAfl-U, dAcl-U, dAbr-U, dAio-U, and dA-U, were synthesized chemically. Characterization of these dimers has been performed by UV absorption, circular dichroism, and proton nuclear magnetic resonance spectroscopy. The results show that the dimers containing 2'-halo-2'-deoxyadenosines have stacked conformations with a geometry similar to that of A-U and the degree of stacking decreases in the order dAfl-U greater than dAcl-U greater than dAbr-U greater than dAio-U. dAcl-U is assumed to have the same degree of stacking as A-U. dA-U takes a more stacked conformation than does dAio-U, but the mode of stacking is different from those of the other dimers. The effects of the 2' substituents on dimer conformation are discussed in terms of electronegativity, molecular size, and hydrophobicity.     

Interaction of three Caenorhabditis elegans isoforms of translation initiation factor eIF4E with mono- and trimethylated mRNA 5' cap analogues

Translation initiation factor eIF4E binds the m(7)G cap of eukaryotic mRNAs and mediates recruitment of mRNA to the ribosome during cap-dependent translation initiation. This event is the rate-limiting step of translation and a major target for translational control. In the nematode Caenorhabditis elegans, about 70% of genes express mRNAs with an unusual cap structure containing m(3)(2,2,7)G, which is poorly recognized by mammalian eIF4E. C. elegans expresses five isoforms of eIF4E (IFE-1, IFE-2, etc.). Three of these (IFE-3, IFE-4 and IFE-5) were investigated by means of spectroscopy and structural modelling based on mouse eIF4E bound to m(7)GDP. Intrinsic fluorescence quenching of Trp residues in the IFEs by iodide ions indicated structural differences between the apo and m(7)G cap bound proteins. Fluorescence quenching by selected cap analogues showed that only IFE-5 forms specific complexes with both m(7)G- and m(3)(2,2,7)G-containing caps (K(as) 2 x 10(6) M(-1) to 7 x 10(6) M(-1)) whereas IFE-3 and IFE-4 discriminated strongly in favor of m(7)G-containing caps. These spectroscopic results quantitatively confirm earlier qualitative data derived from affinity chromatography. The dependence of K(as) on pH indicated optimal cap binding of IFE-3, IFE-4 and IFE-5 at pH 7.2, lower by 0.4 pH units than that of eIF4E from human erythrocytes. These results provide insight into the molecular mechanism of recognition of structurally different caps by the highly homologous IFEs.     

Cooperative stacking and hydrogen bond pairing interactions of fragment peptide in cap binding protein with mRNA cap structure

The stacking and hydrogen bonding abilities of Trp-(Gly)n-Glu (n = 0 approximately 3) for the interaction with 7-methylguanine (m7G) base were examined by fluorescence and 1H-NMR methods, and it was shown that they correlate with the distance between the Trp and Glu residues, and become most significant when both residues are separated from each other by two Gly residues (n = 2). Based on this insight, the sequence conserved between the human and yeast cap binding proteins (CBPs) was surveyed, and the sequence of Trp-Glu-Asp-Glu (No. 102-105 in human CBP) was selected as a probable site for the binding with mRNA cap structure. Thus, the stacking and hydrogen bonding abilities of Trp-Glu-Asp-Glu with m7G cap structure were examined by comparative experiments using its analogous peptides. The results showed that the fourth Glu residue is important not only for the construction of hydrogen bond pairing with m7G base but also for strengthening the stacking interaction between the Trp indole ring and m7G base. Taking account of the recognition analysis using the mutant CBP proteins by site-directed mutagenesis (Ueda, H., Iyo, H., Doi, M., Inoue, M., Ishida, T., Morioka, H., Tanaka, T., Nishikawa, S. and Uesugi, S. (1991) FEBS Lett. 280, 207-210), this cooperative interaction could be important for the recognition of mRNA cap structure.     

Inhibition of eukaryotic translation by nucleoside 5'-monophosphate analogues of mRNA 5'-cap: changes in N7 substituent affect analogue activity

Nucleotide cap analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized in which the 7-methyl moiety was replaced with 7-ethyl (e7), 7-propyl (p7), 7-isopropyl (ip7), 7-butyl (b7), 7-isobutyl (ib7), 7-cyclopentyl (cp7), 7-(carboxymethyl) (cm7), 7-benzyl (bn7), 7-(2-phenylethyl) [7-(2-PhEt)], and 7-(1-phenylethyl) [7-(1-PhEt)]. These derivatives were assayed as competitive inhibitors of capped mRNA translation in reticulocyte lysate. We observed that N7 alkyl and alicyclic substituents larger than ethyl significantly decreased the inhibitory activity of these cap analogues presumably by decreasing their affinity for cap binding proteins, which participate in the initiation of translation. This result defined a maximum size for this class of N7 substituents in the nucleotide binding domain of cap binding proteins. Like m7GMP, the N7-substituted GMP derivatives synthesized in this study were found to be predominantly in the anti conformation as determined by proton NMR analyses. However, bn7GMP and 7-(2-PhEt)GMP, which have aromatic N7 substituents, were more effective than m7GMP as competitive inhibitors of translation. The increased affinity of bn7GMP for cap binding proteins was further examined by synthesis of beta-globin mRNA containing 5'-bn7G, 5'-m7G, or 5'-e7G cap structures. These modified mRNAs were tested as translation templates. Messenger RNA capped with bn7G was observed to increase the translation activity of the template 1.8-fold relative to that of its m7G-capped mRNA counterpart. By contrast, e7G-capped mRNA was 25% less active than m7G-capped mRNA.2+V photo-cross-linking of m7G-capped mRNA to cap binding proteins     

Isolation and characterization of rabbit anti-m3 2,2,7G antibodies.

Antibodies specific for intact 2,2,7-trimethylguanosine (m3 2,2,7G) were induced by immunization of rabbits with a nucleoside-human serum albumen (HSA) conjugate. Competition radioimmunoassay showed that the antibody distinguishes well between intact m3 2,2,7G and its alkali-hydrolysed form (m3 2,2,7G*). Antibody specificity is largely dependent on the presence of all three methyl groups in m3 2,2,7G: none of the less extensively methylated nucleosides m7G, m2G and m2 2,2G is able to compete efficiently with the homologous hapten. Little or no competition was observed with m1G, m1A, m6A, m5U and each of the four unmodified ribonucleosides. Binding studies with nucleoplasmic RNAs from Ehrlich ascites cells suggest that the antibody reacts specifically with the m3 2,2,7G-containing cap structure of the small nuclear U-RNAs (U-snRNAs). Thus the antibody should be a valuable tool for studying the role of the 5'-terminal regions of the U-snRNAs of eucaryotic cells.     

Synthesis, structure and thermodynamic properties of 8-methylguanine-containing oligonucleotides: Z-DNA under physiological salt conditions.

Various oligonucleotides containing 8-methylguanine (m8G) have been synthesized and their structures and thermodynamic properties investigated. Introduction Of M8G into DNA sequences markedly stabilizes the Z conformation under low salt conditions. The hexamer d(CGC[M8G]CG)2 exhibits a CD spectrum characteristic of the Z conformation under physiological salt conditions. The NOE-restrained refinement unequivocally demonstrated that d(CGC[m8G]CG)2 adopts a Z structure with all guanines in the syn conformation. The refined NMR structure is very similar to the Z form crystal structure of d(CGCGCG)2, with a root mean square deviation of 0.6 between the two structures. The contribution of m8G to the stabilization of Z-DNA has been estimated from the mid-point NaCl concentrations for the B-Z transition of various m8G-containing oligomers. The presence of m8G in d(CGC[m8G]CG)2 stabilizes the Z conformation by at least deltaG = -0.8 kcal/mol relative to the unmodified hexamer. The Z conformation was further stabilized by increasing the number of m8Gs incorporated and destabilized by incorporating syn-A or syn-T, found respectively in the (A,T)-containing alternating and non-alternating pyrimidine-purine sequences. The results suggest that the chemically less reactive m8G base is a useful agent for studying molecular interactions of Z-DNA or other DNA structures that incorporate syn-G conformation.     

Charge distribution in 7-methylguanine regarding cation-pi interaction with protein factor eIF4E

Electric charge distribution in mRNA 5' cap terminus has been exhaustively characterized in respect to the affinity for cap-binding proteins. Formation of the stacked configuration of positively charged 7-methylguanine in between two aromatic amino acid rings, known as sandwich cation-pi stacking, is thought to be prerequisite for the specific recognition of the cap by eukaryotic initiation factor eIF4E; i.e., discrimination between the cap and nucleotides without the methyl group at N(7). Nuclear magnetic resonance spectroscopy of (15)N/(13)C-double-labeled 7-methylguanosine 5'-triphosphate and 7-methylguanosine, as well as their unsubstituted counterparts, GTP and guanosine, yielded characteristic changes of the electron-mediated spin-spin couplings and chemical shifts due to the methylation at N(7). The experimentally measured changes of the nuclear magnetic resonance parameters have been analyzed in respect to the electric charge distribution calculated by means of quantum chemical methods, and interpreted in terms of new proposed positive charge localization in the 7-methylguanine five-member ring.     

Insertion of an N7-methylguanine mRNA cap between two coplanar aromatic residues of a cap-binding protein is fast and selective for a positively charged cap

The N7-methylated guanosine (m7G) cap structure, which is found at the 5' ends of mature eukaryotic mRNAs, is critical to a myriad of biological processes. The twenty structures of complexes of cap nucleosides and nucleotides and methylated bases with the vaccinia virus VP39, a cap-specific RNA 2'-O-methyltransferase, which we have determined previously, have revealed the atomic basis of cap binding. The precise insertion and tight fitting of the m7Gua moiety of the cap between two parallel aromatic residues that are spaced only 6.8 A apart governs the high specificity of binding. Here we report the investigation of the reaction mechanism of VP39 with three capped ligands (m7G, m7GpppG, and m7GpppGA3) by fluorescence stopped-flow technique. Cap binding is a simple one-step mechanism with very fast association rate constant (approximately 10(7) M-1 s-1). Moreover, the pH dependence on the association rate constant of m7G binding indicates that only the positively charged keto tautomer of the cap is recognized and bound. The association and dissociation rate constants and affinity constants of the three ligands do not vary greatly, demonstrating that binding is achieved almost entirely by the interactions of m7Gua with two aromatic residues in a cation-pi sandwich.     

Conformation of dinucleoside monophosphates modified with benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide as measured by circular dichroism

The conformational properties of GpU modified with the reactive derivative of benzo[a]pyrene, (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, has been investigated utilizing circular dichroism spectroscopy. Binding of this carcinogen to the N2 of G residues in GpU resulted in the formation of four compounds (I to IV) representing two pairs of diastereoisomers. The molar ellipticity values of the modified dimers were approximately twofold higher than those of the modified guanosine monomers. These values were decreased appreciably when the spectra of the dimers were obtained at 80 degrees C or in methanol rather than at 25 degrees C in water, suggesting that under the latter conditions there is a stacking interaction between the carcinogen and the neighboring uridine residue. Based on these results, a conformation is proposed for modified GpU. It includes insertion of the benzo[a]pyrene moiety, by rotation of the modified guanine residue about its glycoside bond, coplanar to the neighboring uridine and perpendicular to the phosphodiester backbone.     

Antibodies elicited by N2,N2-7-trimethylguanosine react with small nuclear RNAs and ribonucleoproteins

The 5' ends of U1, U2, U3, U4, and U5 small nuclear RNAs (snRNA) are capped by a structure which contains N2,N2-7-trimethylguanosine (m2,2,7 G). m2,2,7 G was used as hapten to raise antibodies in rabbits, and these antibodies were linked to Sepharose. When deproteinized RNA was passed through this antibody column, these snRNA species were retained by the column. Conversely, 4 S, 5 S, 5.8 S, U6, and 7 S RNA, whose 5' termini do not contain m2,2,7 G, were not recognized. After a nuclear extract was loaded on the column, U1 RNA and some U2 RNA were retained. Therefore, the 5' ends of at least U1 RNA are accessible when this RNA species is in small nuclear ribonucleoprotein particle (snRNP) form. This is of interest, since it has been proposed that the 5' terminus sequence of U1 RNA may hybridize with splice junctions in heterogeneous nuclear ribonucleoprotein particles (hnRNP) during mRNA splicing. The retention of m2,2,7 G-containing RNA species by these antibodies is not due to association of snRNAs or snRNPs with heterogeneous nuclear RNA (hnRNA) or hnRNP (and antibody recognition of 7-monomethylguanosine residues in hnRNA), since the reaction still occurs after removal of hnRNA or hnRNP by sucrose gradient centrifugation.     

Structural requirements for the specific recognition of an m7G mRNA cap

7-Methylguanosine (m(7)G), also known as the mRNA "cap", is used as a molecular tag in eukaryotic cells to mark the 5' end of messenger RNAs. The mRNA cap is required for several key events in gene expression in which the m(7)G moiety is specifically recognized by cellular proteins. The configurations of the m(7)G-binding pockets of a cellular (eIF4E) and a viral (VP39) cap-binding protein have been determined by X-ray crystallography. The binding energy has been hypothesized to result from a pi-pi stacking interaction between aromatic residues sandwiching the m(7)G base in addition to hydrogen bonds between the base and acidic protein side chains. To further understand the structural requirements for the specific recognition of an m(7)G mRNA cap, we determined the effects of amino acid substitutions in eIF4E and VP39 cap-binding sites on their affinity for m(7)GDP. The requirements for residues suggested to pi-pi stack and hydrogen bond with the m(7)G base were examined in each protein by measuring their affinities for m(7)GDP by fluorimetry. The results suggest that both eIF4E and VP39 require a complicated pattern of both orientation and identity of the stacking aromatic residues to permit the selective binding of m(7)GDP.     

Structural study of DNA duplex containing an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide frameshift by NMR and restrained molecular dynamics

The presence of an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide (F) residue, a ring fragmentation product of thymine, in a frameshift context in the sequence 5'-d-(AGGACCACG)*d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. Two-dimensional NMR studies show that the formamide residue, whether the cis or trans isomer, is rotated out of the helix and that the bases on either side of the formamide residue in the sequence, G14 and T16, are stacked over each other in a way similar to normal B-DNA. The cis and trans isomers were observed in the ratio 3:2 in solution. Information extracted from 31P NMR data reveal a modification of the phosphodiester backbone conformation at the extrahelical site, which is also observed during the molecular dynamics simulations.     

Ecological Stoichiometry of Microbial Biomass Carbon,Nitrogen and Phosphorus on Bauxite Residue Disposal Areas

Abstract

Ecological succession by microbial activity on bauxite residue disposal areas (BRDAs) would accumulate nutrients and convert the residue into a soil-like material. However, the role of microorganisms in nutrient cycling remains elusive on BRDAs. Carbon (C), nitrogen (N) and phosphorus (P) ecological stoichiometry is a critical indicator of nutrient cycling in an ecosystem. In order to investigate the changes in nutrients following long-term natural weathering process, the contents of C, N, P, microbial biomass carbon (MBC), nitrogen (MBN), and phosphorus (MBP) were measured in chronological stacks of bauxite residue. Deeply, their ecological stoichiometric characteristics were analyzed. Compared to freshly stacked residue, organic carbon (OC), total nitrogen (TN) and available phosphorus (AP) have increased 89%, 1640%, and 369% after 20?years, respectively. The C/N in 20-year-old residue (BR20) is 12.41, which close to the mean range of soil C/N in China. Bauxite residue C/P and N/P increased significantly with the stacking age increased. MBC/MBN decreased from 6.75 to 4.52 after stacked for 5?years, whilst MBC/MBP increased from 23.74 to 59.16 with stacking age. Data analysis of C, N, P and MBC, MBN, MBP in bauxite residue correlated significantly, indicating that microbial biomass can be used as a biological indicator to evaluate bauxite residue quality. This study revealed that BRDAs ecosystem development reaches homeostasis gradually, whilst CNP and MBCNP substrate ratio can be used as an effective tool to explore the mechanism of nutrient cycling.     

Nuclear magnetic resonance studies on yeast tRNAPhe. II. Assignment of the iminoproton resonances of the anticodon and T stem by means of nuclear Overhauser effect experiments at 500 MHz.

Resonances of the water exchangeable iminoprotons of the T and anticodon stem of yeast tRNAPhe were assigned by means of Nuclear Overhauser Effects (NOE's). Together with our previous assignments of iminoproton resonances from the acceptor and D stem (A. Heerschap, C.A.G. Haasnoot and C.W. Hilbers (1982) Nucleic Acids Res. 10, 6981-7000) the present results constitute a complete assignment of all resonances of iminoprotons involved in the secondary structure of yeast tRNAPhe with a reliability and spectral resolution not reached heretofore. Separate identification of the methylprotons in m5C40 and m5C49 was also possible due to specific NOE patterns in the lowfield part of the spectrum. Our experiments indicate that in solution the psi 39 residue in the anticodon stem is orientated in a syn conformation in contrast to the normally observed anti orientation of the uracil base in AU basepairs. Evidence is presented that in solution the acceptor stem is stacked upon the T stem. Furthermore, it turns out that in a similar way the anticodon stem forms a continuous stack with the D stem, but here the m2(2)G26 residue is located between the latter two stems (as is found in the X-ray crystal structure). The stacking of these stems is not strictly dependent on the presence of magnesium ions. NOE experiments show that these structural features are preserved when proceeding from a buffer with magnesium ions to a buffer without magnesium ions although differences in chemical shifts and NOE intensities indicate changes in the conformation of the tRNA.