Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate     

Changes in brain components during the development of mice homozygous for the locus “dwarf” (dw)1,5

Brain composition and developmental changes were investigated in mice homozygous for the locus dwarf, and characterized by a reduced level of growth hormone, thyroid stimulating hormone, and prolactin, and by secondary hypothyroidism. The difference in adult brain weight (–32%) between the dwarf and the normal mice was not found to parallel the difference in body weight (–71%), whereas the differences in the weight of the liver (–79%) and that of the kidney (–75%) did. Several biochemical parameters of brain development were assayed in dwarf and normal mice between the ages of 15 and 210 days. Levels of cerebrosides, sulfatides, gangliosides, phospholipids, cholesterol, protein, and RNA (per gram wet weight) were the same for the dwarf and the controls, but the net difference in total brain DNA was less than the net total brain RNA difference (–11% vs. –27%). Total brain lipids (absolute quantities) were the same at 15 days. The difference was –37% by the 50th day, and remained constant thereafter. No change in the specific activity of 2,3-cyclic nucleotide 3-phosphohydrolase or 3-phosphoadenosine-5-phosphosulfate: galactocerebroside sulfotransferase was observed. These data suggest that the regulation of the development of brain structures is maintained, but the level of the synthesis of the various brain constituents is reduced in proportion to the brain weight. The development of the dwarf brain seems to proceed harmoniously.Abbreviations used PAPS 3-phosphoadenosine-5-phosphosulfate - PAPS-CST 3-phosphoadenosine-5-phosphosulfate:galactocerebroside sulfotransferase - CNP 2, 3-cyclic nucleotide 3-phosphohydrolase - Neu NAc N-acetylneuraminic acid This paper is part of the Doctorat d'Etat thesis of L. L. Sarliève.     

Sensitive 1H-31P correlations with 5′ methylene protons of DNA via homonuclear double-quantum coherence

A novel pulse sequence is presented for the correlation of 5 and 5 protons in DNA with phosphorus. Double-quantum coherence between the methylene protons is used to generate ¹H5-³¹P and ¹H5-³¹P cross peaks in an HMQC-type experiment. The resolution for these cross peaks is significantly improved over that of conventional HSQC experiments, as cross peaks between ¹H4 and ³¹P are largely suppressed and a 3D version of the experiment can be performed with little penalty in sensitivity. In addition, sensitivity is favoured by slower relaxation of the double-quantum coherence and a more favourable multiplet fine structure in the acquisition dimension.     

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

Summary In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3-position. Similar to the flavonoid 3-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3-position in Petunia. Thus, the introduction of the 3-hydroxyl group is clearly achieved by hydroxylation of C₁₅-intermediates, and the concomitant occurrence of the 3,4-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.     

Sugar conformation of a stereospecific 2′-R or 2′-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants

The sugar conformation of a DNA decamer was studied with proton-proton ³J coupling constants. Two samples, one comprising stereospecifically labeled 2-R-²H for all residues and the other 2-S-²H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980–6986; Nucleosides Nucleotides (1995) 14, 333–336], the deuterium labeling being highly stereospecific 99% for all 2-²H, 98% for 2-²H of A, C, and T, and 93% for 2-²H of G). The ³J values of all H1-H2 and H1-H2 pairs, and several H2-H3 and H2-H3 pairs were determined by line fitting of 1D spectra with 0.1–0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3-exo and C2-endo with _m values of 26° to 44°, except for the second and 3 terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from J_H1H2 and J_H1H2 values. For C10, the N–S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1-exo conformation with 27° distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.     

The formation of peptides from the 2′(3′)-glycyl ester of a nucleotide

Summary We have synthesized 2(3)-O-(glycyl)-adenosine-5-(O-methylphos-phate), an analogue of the 3-terminus of aminoacylated tRNA. A 0.4M solution of this compound maintained at pH 8.2, yields 5.5% of diglycine and 11.5% of diketopiperazine, in addition to the hydrolysis products glycine and adenosine-5-(O-methylphosphate). Under the same conditions, glycine ethyl ester reacts much more slowly, but ultimately gives similar yields of diglycine and diketopiperazine.The aminolysis of 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by free glycine is relatively inefficient, but serine reacts 20 times more rapidly and yields up to 50% of N-glycylserine. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-gly 2,3-O-(bis-glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - gly Et glycine ethyl ester - gly-ser N-glycylserine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - Boc-gly N-tert-butyloxycarbonylglycine - MepA-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-Boc-gly 2,3-O-(bis-Boc-glycyl)-adenosine-5(O-methylphosphate) - (gly)₂ diglycine - (gly)₃ triglycine     

Geometry of the dry-state oligomerization of 2′,3′-cyclic phosphates

Summary Evaporation of a solution of thymidine plus either theexo or theendo diastereomer of uridine cyclic 2,3-O, O-phosphorothioate (U > p(S) in 1,2-diaminoethane hydrochloride buffer gave the 2,5 and 3,5 isomers of (P-thio) uridylylthymidine (Up(S)dT) in a ratio of 1:2 with a combined yield of about 20%. These isomers were re-converted to U > p(S) and dT by a reaction that is known to proceed by an in-line mechanism. Both the 2,5 and 3,5 isomers gave as product the same diastereomer of U > p(S) that had been used originally in their formation. These dry-state prebiotic reactions (Verlander, Lohrmann, and Orgel 1973) are thus shown to be stereospecific, and both the 2,5 and 3,5 internucleotide bonds are formed by an in-line mechanism.Abbreviations DAE 1,2-diaminoethane - HPLC high pressure liquid chromatography - RNase bovine pancreatic ribonuclease A, EC 3.1.4.22 - TEAB triethylammonium bicarbonate - tris tris(hydroxymethyl)aminomethane - UMP(S) uridine monophosphorothioate - U > p uridine cyclic 2,3-phosphate - U > p(S) uridine cyclic 2,3-O, O-phosphorothioate - Up(S)dT (P-thio)uridylylthymidine - U2p(R_p-S)5dT (P-thio)uridylylthymidine with theR configuration at phosphorous, and a 2,5 internucleotide linkage     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Very slow growth of Bacillus polymyxa: Stringent response and maintenance energy

Bacillus polymyxa grown in a recycling fermentor shows the same behavior previously observed with Escherichia coli: 3 successive growth phases. In the last 2 phases the growth rate is linear and the apparent maintenance energy demand rate and the molar growth yield are both independent of the specific growth rate, , and of the cells mass. The final phase of very slow growth is an indefinitely prolonged state of strong, stringent control, the regulatory system based on guanosine 3-diphosphate 5-diphosphate, and guanosine 3-diphosphate 5-triphosphate. The maximum cost of this stringent response is calculated to be 9% of the energy available to these energy-limited cells. There is a further energy cost contained in substantial amounts of DNA, RNA, and protein released from the cells during the latter 2 growth phases. The cost of production of these extra cellular anabolites ranges from 8–11% of the available energy.After a carbon-energy upshift in phase 3, the population growth rate immediately returned to that of early phase 2 growth, 50 h or more earlier.If maintenance energy is considered as energy expended by cells to maintain homeostasis, catabolic capacity, or anabolic potential, then the cost of stringent control — which preserves the fidelity of protein synthesis in slowly growing cells — must be considered a maintenance energy cost.Abbreviations GPR glucose provision rate - F_R medium flow rate - S_R substrate concentration - V_F fermentor volume - F_S filtrate removal rate - ppGpp guanosine 3-diphosphate 5-diphosphate - pppGpp guanosine 3-diphosphate 5-triphosphate     

Cytochemical studies on the localization of 5′-nucleotidase in the acinar cells of the rat salivary glands

Summary The cytochemical localization of 5-nucleotidase (5-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5-AMPase and non-specific alkaline phosphatase (-GPase) possessed different pH optima.The cytochemical distribution of the reaction products from the 5-AMPase activity was distinct from those of -GPase. 5-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. -GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5-AMPase and -GPase.The results indicate that 5-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.     

Integration of biochemical functions of different cells of RAT gastric mucosa for hydrochloric acid secretion

Summary The regulation patterns of gastric acid secretion in rats were investigated. Pentagastrin and histamine stimulate gastric acid secretion, but the inhibitors of DNA-dependent synthesis of RNA and of proteins prevent only the pentagastrin action. It has been found that pentagastrin induces histidine decarboxylase in gastric mucosa, ensuring local accumulation of histamine. The latter activates adenylate cyclase and results in 3,5-AMP accumulation in gastric tissues. The administration of pentagastrin, histamine or 3,5-AMP enhances the activity of gastric carbonic anhydrase, the enzyme which takes part in HCI formation. The data suggest that these three compounds act sequentially (pentagastrin histamine 3,5-AMP) and the effect of the last one could be mediated through 3,5-AMP dependent protein kinase. The experiments in vitro demonstrated that gastric carbonic anhydrase can be separated into two isoenzymes and the phosphorylation of one of them by the 3,5-AMP dependent protein kinase sharply increases its activity. The findings raise the possibility that histamine and 3,5-AMP, mediating gastrin action, form together with enzymes (histidine decarboxylase, adenylate cyclase, protein kinase, carbonic anhydrase) a cascade of amplifiers.Autoradiographic studies have shown that [³H]-pentagastrin is not bound by oxyntic cells but adheres preferentially to histamine-producing-like endocrine cells and to the chief cells, while³H-histamine adheres preferentially to oxyntic and to chief cells. Electron microscopy indicates that only pentagastrin (but not histamine) initiates in-like endocrine cells ultrastructural changes characteristic for induction. Pentagastrin, histamine and 3,5-AMP administration produces in oxyntic cells ultrastructural changes typical for the secretion processes.These results lead to assumption that pentagastrin (gastrin) induces histidine decarboxylase in-like endocrine cells of gastric glands. Histamine which is secreted enhances adenylate cyclase activity in the neighbouring oxyntic cells where 3,5-AMP dependent protein kinase activates carbonic anhydrase by means of phosphorylation. These different cells form, probably, a multicellular functional unit for gastric acid secretion.An invited article.     

A novel PH-CT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. Application to HIV-2 TAR RNA

A quantitative analysis of J_PH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal ³J_PH3, ³J_PH5 and ³J_PH5 scalar couplings can be obtained by monitoring the intensity decay of the P_i-H3_{i – 1} peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two ³J_PH5 and ³J_PH5 scalar couplings even in the presence of overlapped H5/H5 resonances, since the quantitative information is extracted from the intensity decay of the P-H3 peak. Moreover, the relaxation of the H3 proton is considerably slower than that of the H5/H5 geminal protons and the commonly populated conformations of the phosphate backbone are associated with large ³J_PH3 couplings and relatively small ³J_{PH5 / H5}. These two facts lead to optimal signal-to-noise ratio for the P-H3 correlation compared to the P-H5/H5 correlation.The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10–15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (D_PH) as well. We will present qualitative results for the measurement of P-H_base and P-H2 dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the D_PHs for the 30mer RNA.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

Effect of adenosine monophosphates on the flowering of Impatients balsamina L., a qualitative short-day plant

Summary Adenosine monophosphates (AMPs) cause the induction of floral buds in Impatients balsamina L. under strictly non-inductive photoperiods and hasten it under inductive photoperiods, cyclic AMP being more effective than 3- or 5-AMPs in this regard.Abbreviations cyclic AMP cyclic 3,5-adenosine monophosphate     

Two-dimensional 1H NMR study of two cyclobutane type photodimers of thymidylyl-(3′→5′)-thymidine

2D NMR spectroscopy and J coupling constant analysis are applied to resolve the structure of two photoproducts of thymidylyl-(35)-thymidine. These products are cyclobutane type thymine dimers possessing the cis-syn (the predominant one) and trans-syn geometry. The cis-syn is formed in an ANTI-ANTI conformation about the N-glycosyl linkages and resembles the normal base-stacked configuration. The glycosidic conformation in solution of the 5 terminal fragment differs from the crystal in which the less common SYN conformation is observed. In this isomer only the sugar pucker of the 3 terminal fragment is changed substantially with respect to the dinucleotide. The trans-syn isomer is formed in a SYN-ANTI glycosidic conformation. In this isomer the sugar puckers of both deoxyribose rings are affected and a preference for a pure 2-endo conformation is observed.Abbreviations dTpdT 2-deoxythymidylyl-(35)-2-deoxythymidine - dTp[]dT cyclobutane type photodimers of dTpdT - dTp- and dTp[]- their 5' terminal fragments (fragment A) - -pdT and-[]pdT their 3 terminal fragments (fragment B) - RP-HPLC reversed-phase high-performance liquid chromatography - COSY two-dimensional correlated spectroscopy - 2D NOE two-dimensional nuclear Overhauser spectroscopy     

Pre-moult foraging trips and moult locations of Emperor penguins at the Mawson Coast

The movements of nine breeding adult emperor penguins Aptenodytes forsteri from two colonies, Auster (67° 23S 64° 04E) and Taylor Glacier (67° 28S 60° 54E), were determined by satellite telemetry on their pre-moult foraging trips. While preparing for their annual moult the penguins travelled for 22–38 days and reached distances of up to 618 km from the colony. Six of the nine tracked penguins were followed to three different moult locations all to the west of their breeding colonies and near other known emperor penguins colonies, such as Kloa Point (66°38S, 59°23E) and Fold Island (67°17S, 59°23E). Sea-ice conditions changed throughout the tracking period; as the birds travelled north the sea-ice contracted south.     

5′-AMP hydrolysis by suspensions and homogenates of pancreatic islet cells from normal and cortisone-treated rats

Summary Suspensions of endocrine pancreas cells were prepared by shaking collagenase-isolated rat islets of Langerhans in calcium-free buffer. When incubated with 1.0 mM substrate at pH 7.4, the cells split,P _i from 5-AMP at a rate of 87 nmol/h per g DNA, and from-glycerophosphate at a rate of 25 nmol/h per g DNAK _m for 5 AMP was about 54 M. Adenosine or theophylline inhibited the 5-AMP hydrolysis. Homogenization of the cells increased the activity toward 5-AMP by 23% and that toward-glycerophosphate by 115%. Injecting rats with cortisone had no effect on the 5-AMP hydrolysis by whole cells but significantly increased the activity in cell homogenates; the intracellular activity toward 5-AMP was more than doubled by the cortisone treatment. Staining whole islet cells for 5-AMP-splitting activity resulted in a demarcation of the cell periphery in control rats. Cells from cortisone-treated rats showed heavier deposits of reaction product, and their cell periphery did not stand out as clearly. It is suggested that 5-nucleotidase is largely an ectoenzyme in normal rat islet cells. The cells also contain an as yet unidentified intracellular phosphatase that seems to be solely responsible for the increased hydrolysis of 5-AMP in cortisone-treated rats.     

Chemoenzymatic galactosialylation with integrated cofactor regeneration

As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac (2-8)Gal(1-4)GlcNAc(1-O)-pent-4-ene was synthesized starting from GlcNAc(1-O)-pent-4-ene, UDP-glucose andN-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and (2-6)sialyl-transferase in a complete cofactor regeneration system.Abbreviations Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5-monophosphosialate - CMP cytidine 5-monophosphate - CDP cytidine 5-diphosphate - CTP cytidine 5-triphosphate - Gal galactose - GlcNAc N-acetylglucosamine - UDP uridine 5-diphosphate - UDP-Glc uridine-5-diphosphoglucose - UDP-Gal uridine-5-diphosphogalactose - PEP phosphoenolpyruvate     

Rapid plant regeneration of pea using thidiazuron

A simple and rapid pea regeneration procedure was developed. An average of up to 20 shoots formed from hypocotyl explants of cvs. Sugar Ann and Patriot cultured on Murashige and Skoog basal medium supplemented with 0.5 or 1.0 M thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea). Hypocotyls of Puget and Sugar Daddy did not respond. Regenerated shoots rooted rapidly when cultured on Murashige and Skoog basal medium containing either 2.0 M -naphthaleneacetic acid or 1.0–2.0 M indole-3-butyric acid. Seeds were harvested from regenerated plants after only 9–11 weeks.Abbreviations BAP 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy acetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea)     

Binding patterns and kinetics of RNase a interaction with RNA

Kinetics and binding studies of RNase A and its natural polymeric substrate (RNA), as well as the natural mixture of free 3-ribonucleotides, were performed by difference spectrophotometry. The obtained kinetic saturation curve, with an anomalous nonhyperbolic shape and a distinct transition point, showed the interchange between the two conformational forms of the enzyme. This occurred in a narrow range of substrate concentration. At low substrate concentration, in spite of the existence of one catalytic cleft, RNase A behaves as a cooperative system, perhaps due to the interactions among the four cooperative binding subsites in the active cleft. At high substrate concentration, the conformational change did occur and was accompanied by a decrease in cooperativity and increment of the catalytic constant. The multiphasic shape of the binding curve, which, in the presence of the enzyme, produced 3-ribonucleotides (as the ligand molecules), shows four binding subsites. The first three subsites are specific for the attachment of phosphate, ribose, and base moieties belonging to the first bound 3-ribonucleotide in the direction of 3-phosphate ribose base-5. The fourth subsite relates to the second phosphate group of the second bound 3-ribonucleotide. The binding direction also converts to 5-phosphate ribose base-3 for the ribonucleotide monomers in the RNA structure.