Branchpoint Sugar Stereochemistry Determines the Hydrolytic Susceptibility of Branched RNA Fragments by the Yeast Debranching Enzyme (YDBR)

Abstract

A series of branched RNAs (Y-shaped) related to yeast pre-mRNA splicing intermediates were synthesized incorporating both natural (i.e., ribose) and non-natural (i.e., arabinose, xylose and acyclic nucleoside) branchpoints in order to examine the effect of sugar conformation and phosphodiester configuration on yDBR hydrolytic efficiency. The results indicate that 2′-phosphodiester scission with yDBR occurs only with a cis-arrangement of phosphate groups at the branchpoint (i.e., ribose) thereby discriminating between all other configurations.     

Determination of 2′-O-methyl groups in 32P-labeled RNAs

A two-dimensional method for the detection of ribose methyl groups in³²P-labeled RNAs is described which is suitable for both qualitative and quantitative purposes. The method involves fractionation of an alkaline hydrolysate of the RNA by two-dimensional electrophoresis of the digest on DEAE-cellulose paper; prior to the second electrophoresis the products are dephosphorylated on the paper by alkaline phosphatase. After visualization of the ribose-methylated (i.e., alkali-stable) oligonucleotides by radioautography, quantitation of the number of ribose methyl groups is possible since each ribose methyl group corresponds to one phosphate group. When applied to a bacterial 23S rRNA the method proved to be accurate enough for determination of the low level (0.1%) of ribose-methylated nucleosides in this RNA. It could also be demonstrated by this sensitive method that the RNA from the small ribosomal subunit of yeast mitochondria is devoid of ribose methyl groups.     

Synthesis and purine receptor affinity of 6-oxopurine nucleosides and nucleotides containing (N)-methanocarba-pseudoribose rings

6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopropane and cyclopentane rings in place of the ribose) were synthesized and the adenosine receptor affinity measured. Guanine or hypoxanthine was coupled at the 7-position, or 1,3-dibutylxanthine was coupled at the 9-position. The pseudoribose ring was also substituted at the 5'-position with an N-methyluronamide or with phosphate groups.     

Synthesis, characterization, and biological properties of small branched RNA fragments containing chiral (Rp and Sp) 2',5'-phosphorothioate linkages

Synthetic branched RNA fragments were prepared to examine the stereochemical requirements for hydrolysis of RNA lariats by the yeast debranching enzyme (yDBR). Specifically, two branched trinucleoside diphosphates and a tetranucleoside triphosphate containing a 2',5'-linked phosphorothioate linkage of defined stereochemistry, namely Rp-A(2'ps5'G)pC, Sp-A(2'ps5'G)pC and Sp-ApA(2'ps5'G)pC, were prepared via solution-phase methods. Unlike the all-phosphodiester control, A(2'p5'G)pC, the Rp-thioated trimer was not cleaved by yDBR, demonstrating that changing the pro-Rp oxygen at the 2',5' phosphodiester bond averts hydrolysis by the enzyme. In contrast, the Sp branched compounds (trimer and tetramer) were cleaved yDBR, albeit with reduced efficiency relative to the corresponding all-phosphodiester branched compounds. Furthermore, the small branched RNAs (5 nt) were not cleaved as efficiently as a 18-nt bRNA, suggesting that the enzyme appears to have a stronger preference for larger bRNA substrates. The non-hydrolyzable branched RNA fragments prepared during these studies may be promising candidates for the future co-crystallization and X-ray analyses of DBR:bRNA complexes.     

Methylglyoxal-induced glycation affects protein topography

Methylglyoxal is a metabolic byproduct that is elevated in diabetic tissue. We examined the effects of methylglyoxal on cytosolic aspartate aminotransferase (cAAT), which is an enzyme previously shown to be modified by glyceraldehyde, acrolein, and ribose 5-phosphate. In the present study we observed that methylglyoxal caused real-time changes in tryptophan (intrinsic) fluorescence. Millimolar concentrations of methylglyoxal predominately decreased the fluorescence emission at 388 nm. While micromolar concentrations also decreased emission at 388 nm, low levels of methylglyoxal caused a prominent redshift in the wavelength of maximal emission. The changes in intrinsic fluorescence reflect definable changes in protein topography. These observations are consistent with a change in conformation that is more compact than that of native cAAT, suggesting that intramolecular cross-links (i.e., lysine-lysine) or hydrophobic pockets (i.e., carboxyethyl-lysines) were formed. Methylglyoxal also inhibited activity, and the inhibition correlated with the methylglyoxal-induced change in protein conformation.     

Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase

A steady state kinetic investigation of the P(i) activation of 5-phospho-d-ribosyl alpha-1-diphosphate synthase from Escherichia coli suggests that P(i) can bind randomly to the enzyme either before or after an ordered addition of free Mg(2+) and substrates. Unsaturation with ribose 5-phosphate increased the apparent cooperativity of P(i) activation. At unsaturating P(i) concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with P(i) directs the subsequent ordered binding of Mg(2+) and substrates via a fast pathway, whereas saturation with ribose 5-phosphate leads to the binding of Mg(2+) and substrates via a slow pathway where P(i) binds to the enzyme last. The random mechanism for P(i) binding was further supported by studies with competitive inhibitors of Mg(2+), MgATP, and ribose 5-phosphate that all appeared noncompetitive when varying P(i) at either saturating or unsaturating ribose 5-phosphate concentrations. Furthermore, none of the inhibitors induced inhibition at increasing P(i) concentrations. Results from ADP inhibition of P(i) activation suggest that these effectors compete for binding to a common regulatory site.     

Ribose modified nucleosides and nucleotides as ligands for purine receptors

Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3',5'-bisphosphate derivatives act as selective P2Y1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y1 and A1 and A3ARs.     

Cooperative interactions in single-strand oligomers of adenylic acid

Optical rotatory dispersion measurements were made on the oligonucleotides (pA)₂, (pA)₄, and (pA)₆ at neutral pH over the temperature range 5–85°C., and compared to similar data for polyriboadenylic acid. The data were interpreted in terms of a temperature-dependent stacking of the bases in the single-strand oligomers, with very little dependence of the degree of stacking on the chain length. These results can be explained by a theory of cooperative stacking. The degrees of freedom available per residue are rotations about the five backbone covalent bonds and the bond connecting the base to the ribose ring. To nucleate a stacking interaction between neighboring bases the backbone sequence must be ordered as must be the two bases. For this stack to grow by one base a backbone sequence must again be ordered, but only one additional base must be ordered. Thus, the degree of freedom of the base with respect to the ribose ring determines the extent of the cooperative effect and hence the effect of chain length. A matrix formulation of the partition function is presented which incorporates this cooperative nature of the interaction and is shown to be in fair agreement with the data. The entropy of ordering a base with respect to the ribose ring is found to be 0.68 e.u., which suggests that the purine has a torsional oscillation when unstacked, but does not have several isoenergetic positions of internal rotation available. The enthalpy of stacking is found to be ?6.5 kcal./mole. A model involving neighbor and next-nearest neighbor interactions could also account for the data. For all practical purposes, the stacking interactions of successive residues can be treated as independent, i.e., the state of one residue is essentially independent of the state of stacking of its neighbors.     

Preference for Ribose Over Deoxyribose in Loop-Closing Base Pairs of Extra Stable Nucleic Acid Hairpins

We have investigated the effect of switching ribose to deoxyribose at the closing base-pair of an extra-stable RNA hairpin. Specifically, we studied the sequence 5′-GGAC(UUCG)GUCC, a dodecanucleotide that folds into a well-characterized, “extra stable” RNA hairpin structure. Recently, we showed that hairpins containing a 2′,5′-linked (UUCG) loop instead of the native 3′,5′-linked loop also exhibit extra-stability (Hannoush and Damha, J. Am. Chem. Soc., 2001, 123, 12368–12374). In this article, we show that the ribose units located at the loop-closing positions (i.e., rC ₄ and rG ₉ ) contribute significantly to the stabilization of RNA hairpins, particularly those containing the 3′,5′-UUCG loop. Interestingly, the requirement of rC4 and rG9 is more relaxed for DNA hairpins containing the 2′,5′-UUCG loop and, in fact, they may be replaced altogether (ribose → deoxyribose) without affecting stability. The results broaden our understanding of the behavior of highly stable (UUCG) hairpin loops and how they respond to structural perturbation of the loop-closing base pairs.     

Uracil salvage pathway in PC12 cells

The salvage anabolism of uracil to pyrimidine ribonucleosides and ribonucleotides was investigated in PC12 cells. Pyrimidine base phosphoribosyl transferase is absent in PC12 cells. As a consequence any uracil or cytosine salvage must be a 5-phosphoribosyl 1-pyrophosphate-independent process. When PC12 cell extracts were incubated with ribose 1-phosphate, ATP and uracil they can readily catalyze the synthesis of uracil nucleotides, through a salvage pathway in which the ribose moiety of ribose 1-phosphate is transferred to uracil via uridine phosphorylase (acting anabolically), with subsequent uridine phosphorylation. This pathway is similar to that previously described by us in rat liver and brain extracts (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273; Mascia et al., Biochim. Biophys. Acta 1472 (1999) 93). We show using intact PC12 cells that they can readily take up uracil from the external medium. The analysis of intracellular metabolites reveals that uracil taken up is salvaged into uracil nucleotides, with uridine as an intermediate. We propose that the ribose 1-phosphate-dependent uracil salvage shown by our in vitro studies, using tissues or cellular extracts, might also be operative in intact cells. Our results must be taken into consideration for the comprehension of novel chemotherapeutics' influence on pyrimidine neuronal metabolism.     

Preference for ribose over deoxyribose in loop-closing base pairs of extra stable nucleic acid hairpins

We have investigated the effect of switching ribose to deoxyribose at the closing base-pair of an extra-stable RNA hairpin. Specifically, we studied the sequence 5'-GGAC(UUCG)GUCC, a dodecanucleotide that folds into a well-characterized, "extra stable" RNA hairpin structure. Recently, we showed that hairpins containing a 2',5'-linked (UUCG) loop instead of the native 3',5'-linked loop also exhibit extra-stability (Hannoush and Damha, J. Am. Chem. Soc., 2001, 123, 12368-12374). In this article, we show that the ribose units located at the loop-closing positions (i.e., rC4 and rG9) contribute significantly to the stabilization of RNA hairpins, particularly those containing the 3',5'-UUCG loop. Interestingly, the requirement of rC4 and rG9 is more relaxed for DNA hairpins containing the 2',5'-UUCC loop and, in fact, they may be replaced altogether (ribose--> deoxyribose) without affecting stability. The results broaden our understanding of the behavior of highly stable (UUCG) hairpin loops and how they respond to structural perturbation of the loop-closing base pairs.     

RNA DNA discrimination by the antitermination protein NusB

The regulation of ribosomal RNA biosynthesis in Escherichia coli by antitermination requires binding of NusB protein to a dodecamer sequence designated boxA on the nascent RNA. The affinity of NusB protein for boxA RNA exceeds that for the homologous DNA segment by more than three orders of magnitude as shown by surface plasmon resonance measurements. DNA RNA discrimination by NusB protein was shown to involve methyl groups (i.e. discrimination of uracil versus thymine) and 2' hydroxyl groups (i.e. discrimination of ribose versus deoxyribose side-chains) in the RNA motif. Ligand perturbation experiments monitored by 1H15N correlation NMR experiments identified amide NH groups whose chemical shifts are affected selectively by ribose/deoxyribose exchange in the 5' and the central part of the dodecameric boxA motif respectively. The impact of structural modification of the boxA motif on the affinity for NusB protein as observed by 1H15N heterocorrelation was analysed by a generic algorithm.     

Initiation of poly(ADP-ribosyl) histone synthesis by poly(ADP-ribose) synthetase

Initiation of poly(ADP-ribosyl) histone synthesis was achieved in vitro using an apparently homogeneous preparation of poly(ADP-ribose) synthetase. When poly(ADP-ribose) was synthesized in the presence of DNA and increase amounts of histone H1, increasing portions (up to about 55%) of the product were found associated with the histone, judging from solubility in 5% HClO4 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Most of the polymers were directly attached to the histone protein and not produced by elongation from pre-existing ADP-ribose; the cohesive end of poly(ADP-ribose), isolated as ribose 5-phosphate with snake venom phosphodiesterase digestion, was labeled almost quantitatively with [ribose (NMN)-14C]NAD. The poly(ADP-ribose) . histone linkage was labile in mild alkali and neutral NH2OH, suggesting that the same bond, probably ester, was formed in this system as in crude chromatin or isolated nuclei. Elongation of a histone-bound monomer into a polymer by this enzyme was previously demonstrated (Ueda, K., Kawaichi, M., Okayama, H., and Hayaishi, O. (1979) J. Biol. Chem. 254, 679-687), but initiation of ADP-ribose chains on histone has never been shown with a purified enzyme. This appeared to be due to the low concentrations of histone so far used. These findings indicated that a single enzyme catalyzes two different types of reaction, i.e. an attachment of ADP-ribose to histone and its elongation into a polymer.     

The formation of a 1-5 phosphodiester linkage in the spontaneous breakdown of 5-phosphoribosyl-alpha-1-pyrophosphate

The decomposition of 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) in the presence of Mg2+ at pH=7.8 yields a combination of products including ribose 5-phosphate, ribose 1-phosphate, 5-phosphoribosyl 1,2 cyclic phosphate, inorganic phosphate, and pyrophosphate. Hydrogen decoupled 31P NMR analysis of the product mixture also exhibits a sharp peak (+2.6 ppm from phosphocreatine) in a chemical shift region which includes phosphodiester bonds. Alkaline phosphatase treatment of the product mixture results in cleavage of monophosphate esters such as ribose 1-phosphate and ribose 5-phosphate, but does not affect the unidentified peak. Homonuclear (1H) correlation spectroscopy (COSY) of a partially purified sample was successful in identifying the hydrogen spectra of this compound. Combined with results from the splitting patterns of selectively decoupled 31P spectra, the COSY data indicate that several hydrogens are directly coupled to the unknown phosphate group with J value matches to the hydrogen on carbon one and to the two hydrogens on carbon five. Heteronuclear (1H-31P) chemical shift correlation studies confirm these couplings and further substantiate the formation of a ribose 1-5 phosphate linkage during the degradation of PRPP under these conditions. It is presently unknown whether this is an intramolecular or intermolecular phosphodiester linkage, although some spectroscopic evidence suggest the intramolecular bond formation, i.e. a ribose 1,5-cyclic phosphate (R-1,5cP). The formation of R-1,5cP helps explain the observation that the 5-phosphate group from PRPP becomes labile during the spontaneous degradation of PRPP.     

Plausible prebiotic synthesis of aldopentoses from simple substrates, glycolaldehyde and formaldehyde

Possible ways of abiotic catalytic synthesis of biologically significant aldopentoses (ribose, xylose, arabinose, lyxose) from elementary substrates, i.e., formaldehyde (FA) and glycolaldehyde (GA) in aqueous solutions are discussed. Conditions in which the process of synthesis of pentoses yields up to 65% relative to the initial concentration of GA: homogeneous borate catalyst NaOH + H₃BO₃, pH 9, [GA]₀ 5 mM, [FA]₀ 50–100 mM have been found.     

Crystal structure of the S-adenosylmethionine synthetase ternary complex: a novel catalytic mechanism of S-adenosylmethionine synthesis from ATP and Met

S-Adenosylmethionine synthetase (MAT) catalyzes formation of S-adenosylmethionine (SAM) from ATP and l-methionine (Met) and hydrolysis of tripolyphosphate to PP(i) and P(i). Escherichia coli MAT (eMAT) has been crystallized with the ATP analogue AMPPNP and Met, and the crystal structure has been determined at 2.5 A resolution. eMAT is a dimer of dimers and has a 222 symmetry. Each active site contains the products SAM and PPNP. A modeling study indicates that the substrates (AMPPNP and Met) can bind at the same sites as the products, and only a small conformation change of the ribose ring is needed for conversion of the substrates to the products. On the basis of the ternary complex structure and a modeling study, a novel catalytic mechanism of SAM formation is proposed. In the mechanism, neutral His14 acts as an acid to cleave the C5'-O5' bond of ATP while simultaneously a change in the ribose ring conformation from C4'-exo to C3'-endo occurs, and the S of Met makes a nucleophilic attack on the C5' to form SAM. All essential amino acid residues for substrate binding found in eMAT are conserved in the rat liver enzyme, indicating that the bacterial and mammalian enzymes have the same catalytic mechanism. However, a catalytic mechanism proposed recently by González et al. based on the structures of three ternary complexes of rat liver MAT [González, B., Pajares, M. A., Hermoso, J. A., Guillerm, D., Guillerm, G., and Sanz-Aparicio. J. (2003) J. Mol. Biol. 331, 407] is substantially different from our mechanism.     

Synthesis and properties of a conformationally restricted spin-labeled analog of ATP and its interaction with myosin and skeletal muscle.

The synthesis is described of a spin-labeled analog of ATP, 2',3'-O-(1-oxy-2,2,6,6-tetramethyl-4-piperidylidene)adenosine 5'-triphosphate (SL-ATP). The spin-label moiety is attached by two bonds to the ribose ring as a spiroketal and hence has restricted conformational mobility relative to the ribose moiety of ATP. The synthesis proceeds via an acid-catalyzed addition of adenosine 5'-monophosphate to 1-acetoxy-4-methoxy-2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine in acetonitrile. The spiroketal product is pyrophosphorylated, and alkaline hydrolysis with concomitant aerial oxidation gives the required product. The spin-labeled moiety probably takes up two rapidly interconverting conformations with respect to the ribose ring on the basis of the 1H NMR spectra of its precursors and related uridine derivatives [Alessi et al. (1991) J. Chem. Soc., Perkin Trans.1,2243-2247]. SL-ATP is a substrate for myosin and actomyosin with similar kinetic parameters to ATP during triphosphatase activity. SL-ATP supports muscle contraction and permits relaxation of permeabilized rabbit skeletal muscle fibers. SL-ADP is a substrate for yeast 3-phosphoglycerate kinase, thus permitting regeneration of SL-ATP from SL-ADP within muscle fibers. Electron paramagnetic resonance (EPR) studies of SL-ADP bound to myosin filaments and to myofibrils show a degree of nanosecond motion independent of that of the protein, which may be due to conformational flexibility of the ribose moiety of ATP bound to myosin's active site. This nanosecond motion is more restricted in myofibrils than in myosin filaments, suggesting that the binding of actin affects the ribose binding site in myosin. EPR studies on SL-ADP bound to rigor cross-bridges in muscle fiber bundles showed the nucleotide to be highly oriented with respect to the fiber axis.     

RIBOSE MODIFIED NUCLEOSIDES AND NUCLEOTIDES AS LIGANDS FOR PURINE RECEPTORS

Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3′,5′-bisphosphate derivatives act as selective P2Y₁ antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y₁ and A₁ and A₃ARs.