Jacques Ninio

Kinetic amplification of enzyme discrimination.

The dependence of the accuracy of enzymatic systems on the mechanism of the catalyzed reaction is investigated, using a probabilistic approach. Certain mechanisms of reaction, involving a delay in one of the steps act as kinetic amplifiers of molecular discriminations. The relationship between our scheme for a delayed reaction [1] and Hopfields scheme [i] is discussed.

A semi-quantitative treatment of missense and nonsense suppression in the strA and ram ribosomal mutants of Escherichia coli Evaluation of some molecular parameters of translation in vivo☆

Abstract The efficiency of missense and nonsense suppressors is affected in different ways in Escherichia coli strains carrying different kinds of ribosomal mutations: see Gorinis review (1970). This led Gorini (1971) to postulate a “ribosomal screen” able to distinguish between normal and mutant tRNAs. Here we examine the alternative hypothesis, that the results on suppression can be accounted for by minor changes in the kinetics of polypeptide chain elongation in mutant ribosomes. A general kinetic scheme for the attachment of tRNA to the A site of ribosomes is described. It is postulated that the tRNA first makes a “loose” bond with the codon. A second event is required to stabilize binding and to allow transpeptidation. The probability that the second event occurs is related to the time that the tRNA sticks to the codon in the loose binding state. Ribosomal mutations would make the transition from loose to tight binding more probable (ram) or less probable (strA), per collision. A method of analysis is developed that enables one to relate directly the experimental measurements on suppression to molecular parameters. By numerical fitting, it is shown that the following set of conditions can account for the behaviour of the various ribosomal mutants. 1. (a) In the wild-type cell, when a codon becomes associated with its cognate tRNA or release factor, the probability of ensuing chain elongation or termination is very close to unity. The probability of elongation decreases to about one-half in strA1 strains. 2. (b) Loaded nonsense suppressor tRNAs su1, su2 and su3 are present in such amounts that they collide with UAG about as often as the release factor does, but their binding is such that peptide bond formation is not very efficient. 3. (c) In contrast, the low efficiency of two missense suppressors appears related to a relatively low frequency of codon-loaded suppressor collisions, while the association itself may be either stronger (su58) or much weaker (su78) than that of the corresponding normal codon-tRNA association.

Efficient algorithms for folding and comparing nucleic acid sequences

Fast algorithms for analysing sequence data are presented. An algorithm for strict homologies finds all common subsequences of length greater than or equal to 6 in two given sequences. With it, nucleic acid pieces five thousand nucleotides long can be compared in five seconds on CDC 6600. Secondary structure algorithms generate the N most stable secondary structures of an RNA molecule, taking into account all loop contributions, and the formation of all possible base-pairs in stems, including odd pairs (G.G., C.U., etc.). They allow a typical 100-nucleotide sequence to be analysed in 10 seconds. The homology and secondary structure programs are respectively illustrated with a comparison of two phage genomes, and a discussion of Drosophila melanogaster 55 RNA folding.

Prediction of pairing schemes in RNA molecules-loop contributions and energy of wobble and non-wobble pairs

Previously published models for predicting pairing schemes in RNA molecules, when applied to tRNA, give the clover leaf structure in only half the cases. We made a systematic investigation of the predictability of the clover leaf structure under various assumptions concerning the energetic contributions of single and double-stranded regions. We tested 21 different models and variants on a set of 100 tRNA sequences and many other variants on a smaller set of sequences. In our models we allowed not only G.C, A.U and G.U pairing, but also every other pair. Under conditions which are much less restrictive than those of previous attempts, we can nevertheless reach 90 per cent predictability for the clover leaf structure of tRNA. A most surprising and far-reaching result is that we can assign to C.G and C.C pairs binding energies quite close to the energies of G.U pairs, and still predict the clover leaf. The following ranking for non-complementary pairs was obtained : G.U, G.G and C.C, U.U, C.A, A.A and G.A, U.C. The main practical innovation which made possible the improvements in predictability are: i) not counting the stacking of base pairs separated by a bulge loop; ii) making the terminal C.Cs in stems more stable than the terminal A.Us by merely -- 0.7 kcal; iii) replacing the distinction between G.C and A.U-closed loops by a distinction based on the presence of loop-favoring residues; iv) carefully adjusting the energetic balance between the various kinds of loops; v) narrowing the gap between the GC/GC and the GC/AU contributions; vi) using observations on nearest-neighbours in tRNA sequences to refine the contributions of G.U pairs.

Connections between translation, transcription and replication error-rates

The analysis of published data from E coli suggests that in all three processes of translation, transcription, and replication, a minority of errors are produced by sub-classes of error-prone components. These add to the basal level of errors a noise of about 10 to 30%. Each one of the three processes contributes to the noisiness of the two others in a loose manner: a large increase in one error-rate produces a moderate increase in another error-rate. The strongest influence is that of transcription on translation errors. There it is possible that a majority of the misacylation errors are produced during the encounter of a correct amino acyl-tRNA ligase with a mistranscribed tRNA. Extreme mutator mutants are expected to produce a moderate increase in translation errors.

Comparative small-angle X-ray scattering studies on unacylated, acylated and cross-linked Escherichia coli transfer RNAIVal☆

Abstract A small-angle X-ray scattering study has been carried out on Escherichia coli tRNAIVal in three states: unacylated (form I); unacylated and cross-linked after ultraviolet irradiation at 335 nm (form II); acylated and N-blocked with the phenoxyacetyl group (form III). The morphological parameters determined on the three forms are almost identical: same molecular weight, same radius of gyration. Consequently, it appears that the over-all shape of the molecule varies very little at the various stages of its function. Besides, the intensity at “high” angles ( 8 > 0·04 A −1 ) reveals a conspicuous difference between forms I and II on one hand, form III on the other. Similar differences are also observed between acylated and unacylated bulk E. coli tRNA at pH 5; a correlation with Mg2+ concentration is observed as well. A theoretical analysis of different methods of taking into account the displacement of solvent by the solute suggests that the high-angle intensity is quite sensitive to solute-solvent interactions. As a tentative conclusion, it is suggested that upon fixation of the amino acid the tRNA molecule undergoes small structure modifications, which affect the distribution of the charged groups at the outer surface of the molecule; as a consequence the solute-solvent interactions, and probably the binding of magnesium, are altered.

Catalysis by a prebiotic nucleotide analog of histidine

A ribosylated derivative of adenine, N6 ribosyl adenine, likely to have formed under prebiotic synthesis conditions, is shown to be as active as histidine in the model reaction of p-nitrophenyl acetate hydrolysis. This property widens the range of reactions accessible to RNA catalysis.

Heteropolynucleotides as templates for non-enzymatic polymerizations

SummaryWe have studied a number of condensation reactions involving ImpU, ImpT, ImpC, ImpA, ImpG, ImpUpG and ImpCpA as activated nucleotide donors and a variety of homo- and hetero-polynucleotides as templates. We did not obtain any evidence of a template effect with ImpU and ImpT, but observed some condensation of ImpC with GpG on appropriate templates. ImpA and ImpG take part in a number of more or less efficient template-directed reactions, as do ImpUpG and ImpCpA.Our results suggest that, on the primitive Earth, pyrimidine nucleotides could most easily have been incorporated into polymers as constituents of short oligomers, which contained one or more purine nucleotide. The linkage of the product depends strongly on the nature of the substrates; the percentage of the natural 3′-5′-linkage was, in some cases, less than 10% and, in others, as high as 70%. Wobble-pairing was often very effective in promoting condensations, suggesting that transition mutations would have been very frequent in prebiotic polynucleotide replication.

Codon-anticodon recognition: The missing triplet hypothesis

Abstract The purpose of this article is to determine whether the rules for codon-anticodon recognition can roughly be the same as for double-stranded RNA associations or if some special configuration which allows one and only one of the bases to wobble is necessary to account for the presence of inosine in a certain number of anticodons in yeast and the presumed absence of related anticodons. It is proposed that the recognition of triplets in a double helical configuration of the type which seems to occur in the ordered segments of transfer RNA is necessarily ambiguous because of the interactions between non-hydrogen-bonded bases on opposite strands (diagonal interactions of stacking). It is then deduced that the complex patterns of degeneracy in codon-anticodon recognition can occur in a situation which is less unsymmetrical with respect to the three positions of the codon-anticodon association than assumed by the wobble hypothesis, provided that some of the potential anticodons be absent in the cell. Our hypothesis makes predictions of this type: if in the anticodon for alanine in yeast (IGC), inosine is substituted by guanine, the resulting modified tRNA will be recognized, in addition to GCC, by GUC which is a codon for valine.

A new approach to DNA polymerase kinetics.

Abstract Little is known of the detailed mechanisms of the polymerization reactions carried out by RNA and DNA polymerases. Besides technical reasons, there are mathematical difficulties not encountered in traditional enzymology. The product of the reaction after one polymerization step is also the substrate of the next step. A number of polymerases, isolated from various sources, have an exonuclease activity. The chain which is being synthesized may be either elongated or trimmed, and its growth has the character of a random walk. In this case, although the overall reaction scheme is more complex, the experiments are more informative, as every dNTP may be transformed into two distinct products: incorporated, or free dNMP. Having solved some of the mathematical difficulties of the random walk problem, we are able to propose a strategy for the study of the polymerization/excision kinetics. We measure the amount y ( t ) of nucleotide that is polymerized at time t and the amount x ( t ) of nucleoside monophosphate that has accumulated. When d y d x is plotted against the concentration of dNTP, a curve is obtained with a characteristic shape, a straight line in a large number of cases. From there, kinetic constants can be estimated. The analysis is made in terms of four possible kinetic schemes. In the most elementary model there are only two rate constants, one for incorporation and one for excision. This model is a limiting case of all other models. The frayed-unfrayed model of Brutlag & Kornberg (1972), Hopfields kinetic proofreading scheme (Hopfield, 1974), and the delayed-escape scheme (Ninio, 1975) are examined in detail, and we show how the kinetic experiments may in principle distinguish between the schemes. Our approach is illustrated with three experiments in which Escherichia coli DNA polymerase I acts on poly(dC), and poly(dT) · oligo(dA) 10 .