An Approach to Prebiotic Synthesis of α-Oligoribonucleotides and Description of Their Properties: Selective Advantage of β-RNA over α-RNA

Oligomerization of α-adenosine 5′-phosphorimidazolide (α-ImpA) has been done in an aqueous solution using a uranyl-ion catalyst or a poly(U) template as a model process of prebiotic synthesis of RNA with α-glycosidic linkage. α-Oligoriboadenylates up to hexamer were formed from α-ImpA by the uranyl-ion catalyst. 3′-5′ Linkage was mainly formed in the oligomerization. The poly(U) template mediated the oligomerization of α-ImpA, but to a very low extent. The yield and chain length of the resulting α-oligomers were far lower than those of the corresponding β-oligomer formation under the same conditions. Physico-chemical properties of α-oligoriboadenylates are presented along with those of the corresponding β-oligoriboadenylates. The results indicate that β-RNA is more advantageous than α-RNA from the points of their synthesis and properties. Received: 10 February 1997 / Accepted: 31 March 1997     

Effect of Inhibitors on the Montmorillonite Clay-Catalyzed Formation of RNA: Studies on the Reaction Pathway

The Langmuir adsorption isotherms of the phosphoroimidazolides of adenosine (ImpA) and uridine (ImpU), dA^5'ppdA and N⁶, N⁶-dimethyladenine binding on montmorillonite are consistent with their forming a monolayer on the clay surface. This suggests the condensation of ImpA and ImpU to oligomers proceeds on the surface of the clay and not in groups of monomers stacked on the clay surfaces. The binding and reactions of ImpU and ImpA on montmorillonite are blocked by N⁶, N⁶-dimethyladenine and dA^5'ppdA. dA^5'ppdA is a better inhibitor of oligomer formation than N⁶, N⁶-dimethyladenine because both adenine rings of dA^5'ppdA bind to the clay surface and block adjacent catalytic sites. An upper limit of 5–10 × 10¹⁵catalytic sites on 50 mg of clay was estimated from the binding of ImpU and the inhibition of oligomer formation by dA^5'ppdA.     

Linear,cyclic, and polypeptides having the sequence -Asp-ϵAhx-Ser-ϵAhx-His-ϵAhx- as catalysts in the ester hydrolytic reactions

Box-Asp-?Ahx-Ser-?Ahx-His-?Ahx-OEt(Linear-6), cyclo(-Asp-?Ahx-Ser-?Ahx-His-?Ahx-)(Cyclic-6) and poly(-Asp-?Ahx-Ser-?Ahx-His-?Ahx-)(Poly-6) were synthesized, and their catalytic actions in the hydrolysis of PNPA were investigated. Linear-6 was prepared by fragment condensations of three peptides having the sequence -Asp(OBzl)-?Ahx-, -Ser(Bzl)-?Ahx- and -His-?Ahx-, respectively, and subsequent debenzylation. Cyclic-6 and Poly-6 were obtained by cyclization and polymerization, respectively, of H-Asp(OBzl)-?Ahx-Ser(Bzl)-?Ahx-His-?Ahx-OH with DPPA, and subsequent deprotections. The reaction velocities in the hydrolysis of PNPA were all proportional to [E] or [S], and all peptides gave the bell-shaped pH-κ_cat profiles having optima around pH 8.2. The reaction velocity of Cyclic-6 was always larger than that of Linear-6 or Poly-6. The velocities of all reactions increased steadily with rise in temperature, and the Arrhenius' plots from T-k_cat relations suggested that the activation energy for the reaction catalysed by Poly-6 is larger than that by Linear- or Cyclic-6. Brief results for the hydrolysis of other substrates, the solvent isotope effect, and the conformational study with c.d. measurements are also reported.     

Foundations of Vectorial Metabolism and Osmochemistry

Chemical transformations, like osmotic translocations, are transport processes when looked at in detail. In chemiosmotic systems, the pathways of specific ligand conduction are spatially orientated through osmoenzymes and porters in which the actions of chemical group, electron and solute transfer occur as vectorial (or higher tensorial order) diffusion processes down gradients of total potential energy that represent real spatially directed fields of force. Thus, it has been possible to describe classical bag-of-enzymes biochemistry as well as membrane biochemistry in terms of transport. But it would not have been possible to explain biological transport in terms of classical transformational biochemistry or chemistry. The recognition of this conceptual asymmetry in favour of transport has seemed to be upsetting to some biochemists and chemists; and they have resisted the shift towards thinking primarily in terms of the vectorial forces and co-linear displacements of ligands in place of their much less informative scalar products that correspond to the conventional scalar energies. Nevertheless, considerable progress has been made in establishing vectorial metabolism and osmochemistry as acceptable biochemical disciplines embracing transport and metabolism, and bioenergetics has been fundamentally transformed as a result.     

Intramolecular RNA replicase: Possibly the first self-replicating molecule in the RNA world

Although there is more and more evidence suggested the existence of an RNA World during the origin of life, the scenario concerning the origin of the RNA World remains blurry. Usually it is speculated that it originated from a prebiotic nucleotide pool, during which a self-replicating RNA synthesis ribozyme may have emerged as the first ribozyme – the RNA replicase. However, there is yet no ersuasive supposition for the mechanism for the self-favouring feature of the replicase, thus the speculation remains unconvincing. Here we suggest that intramolecular catalysis is a possible solution. Two RNA synthesis ribozymes may be integrated into one RNA molecule, as two functional domains which could catalyze the copy of each other. Thus the RNA molecule could self-replicate and be referred to as “intramolecular replicase“ here. Computational simulation to get insight into the dynamic mechanism of emergence of the intramolecular replicase from a nucleotide pool is valuable and would be included in a following work of our group.     

The Early Phases of Genetic Code Origin: Conjectures on the Evolution of Coded Catalysis

A review of the most significant contributions on the early phases of genetic code origin is presented. After stressing the importance of the key intermediary role played in protein synthesis, by peptidyl-tRNA, which is attributed with a primary function in ancestral catalysis, the general lines leading to the codification of the first amino acids in the genetic code are discussed. This is achieved by means of a model of protoribosome evolution which sees protoribosome as the central organiser of ancestral biosynthesis and the mediator of the encounter between compounds (metabolite-pre-tRNAs) and catalysts (peptidyl-pre-tRNAs). The encounter between peptidyl-pre-tRNA catalysts in protoribosome is favoured by metabolic pre-mRNAs and later resulted (given the high temperature at which this evolution is supposed to have taken place) in the evolution of mRNAs with codons of the type GNS. These mRNAs codified only for those amino acids that the coevolution theory of genetic code origin sees as the precursors of all other amino acids. Some aspects of the model here discussed might be rendered real by the transfer-messenger RNA molecule (tmRNA) which is here considered a molecular fossil of ancestral protein synthesis.     

Montmorillonite,Oligonucleotides, RNA and Origin of Life

Na-montmorillonite prepared from Volclay by the titration method facilitates the self-condensation of ImpA, the 5'-phosphorimidazolide derivative of adenosine. As was shown by AE-HPLC analysis and selective enzymatic hydrolysis of products, oligo(A)s formed in this reaction are 10 monomer units long and contain 67% 3',5'-phosphodiester bonds (Ferris and Ertem, 1992a). Under the same reaction conditions, 5'-phosphorimidazolide derivatives of cytidine, uridine and guanosine also undergo self-condensation producing oligomers containing up to 12-14 monomer units for oligo(C)s to 6 monomer units for oligo(G)s. In oligo(C)s and oligo(U)s, 75-80% of the monomers are linked by 2',5'-phosphodiester bonds. Hexamer and higher oligomers isolated from synthetic oligo(C)s formed by montmorillonite catalysis, which contain both 3',5'- and 2',5'-linkages, serve as catalysts for the non-enzymatic template directed synthesis of oligo(G)s from activated monomer 2-MeImpG, guanosine 5'-phospho-2-methylimidazolide (Ertem and Ferris, 1996). Pentamer and higher oligomers containing exclusively 2',5'-linkages, which were isolated from the synthetic oligo(C)s, also serve as templates and produce oligo(G)s with both 2',5'- and 3',5'-phosphodiester bonds. Kinetic studies on montmorillonite catalyzed elongation rates of oligomers using the computer program SIMFIT demonstrated that the rate constants for the formation of oligo(A)s increased in the order of 2-mer < 3-mer < 4-mer ... < 7-mer (Kawamura and Ferris, 1994). A decameric primer, dA(pdA)8pA bound to montmorillonite was elongated to contain up to 50 monomer units by daily addition of activated monomer ImpA to the reaction mixture (Ferris, Hill and Orgel, 1996). Analysis of dimer fractions formed in the montmorillonite catalyzed reaction of binary and quaternary mixtures of ImpA, ImpC, 2-MeImpG and ImpU suggested that only a limited number of oligomers could have formed on the primitive Earth rather than equal amounts of all possible isomers (Ertem and Ferris, 2000). Formation of phosphodiester bonds between mononucleotides by montmorillonite catalysis is a fascinating discovery, and a significant step forward in efforts to find out how the first RNA-like oligomers might have formed in the course of chemical evolution. However, as has been pointed out in several publications, these systems should be regarded as models rather than a literal representation of prebiotic chemistry (Orgel, 1998; Joyce and Orgel, 1999; Schwartz, 1999).     

Energy, Life, and ATP

The mechanism by which ATP is synthesized during oxidative and photophosphorylation has been elucidated by oxygen exchange and other studies: a novel form of catalysis--termed rotary catalysis--is involved.     

Metal ion-catalysed hydrolysis of ampicillin in microbiological growth media

Anodic stripping voltammetry of bacterial growth medium containing copper(II) and ampicillin shows that Cu(II) is complexed by the antibiotic and that this complex decomposes to give Cu(II) complexes with ligands derived from ampicillin. At pH 7, substantial decomposition of ampicillin occurs over a few minutes, and even the very low levels of Cu(II) in Chelex-extracted medium are able effectively to catalyse the decomposition. The significance of this observation was shown during the screening of an Escherichia coli cosmid library for clones exhibiting increased resistance to Zn(II), Co(II) or Cd(II); the unexpected growth of the ampicillin-sensitive host E. coli strain on Luria-Bertani plates containing ampicillin and any of these metals was attributed to metal ion-catalysed decomposition of ampicillin. The instability of ampicillin (and other beta-lactam antibiotics) to metal ion-catalysed hydrolysis means that great care must be taken to ensure that such reactions do not occur in growth media. Furthermore, it is clear that double selection for resistance to ampicillin and metals such as Cu(II), Zn(II), Co(II) and Cd(II) is impossible.     

Enzymic Preparation of Benzyl β-D-Glucopyranoside

Benzyl β-D-glucopyranoside was prepared by an enzyme-catalysed direct reaction between D-glucose, or better cellobiose, and benzyl alcohol in the presence of a minimum amount of water. The enzyme β-glucosidase was used in the immobilized form (adsorbed onto macroporous polyethylene terephthalate or covalently bound on polyglycidyl methacrylate), enabling multiple application.