Jacques Monod

Genetic regulatory mechanisms in the synthesis of proteins.

The synthesis of enzymes in bacteria follows a double genetic control. The socalled structural genes determine the molecular organization of the proteins. Other, functionally specialized, genetic determinants, called regulator and operator genes, control the rate of protein synthesis through the intermediacy of cytoplasmic components or repressors. The repressors can be either inactivated (induction) or activated (repression) by certain specific metabolites. This system of regulation appears to operate directly at the level of the synthesis by the gene of a shortlived intermediate, or messenger, which becomes associated with the ribosomes where protein synthesis takes place.

Sur la biosynthese de la β-galactosidase (lactase) chez Escherichia coli. La specificite de l'induction

1. 1. The formation of β-galactosidase by E. coli is induced exclusively by substances possessing an intact galactosidic radical. Various inversions or substitutions at one or more positions as well as the suppression of the carbon 6 result in the disappearance of the inductive property. 2. 2. The substances which have the inductive property are not necessarily substrates of the enzyme. Thus certain α-galactosides (melibiose) are inductors yet they are not hydrolysed by β-galactosidase. 3. 3. The inductivity is, in general, independent of the affinity for the affinity for the enzyme. Certain substances (phenyl-β-thiogalactoside) which have a high affinity for β-galactosidase in vitro as in vivo are deprived of the inductive property. 4. 4. The formation of β-galactosidase is inhibited by phenyl-β-D-thiogalactoside, but the inhibition is not competitive, while it should be if it was due to the inhibition of the enzyme. 5. 5. These observations are incompatible with all hypotheses which imply that the induction is connected, either with the activity of the enzyme, or with the formation of a specific complex between the enzyme and the inductor.

Kinetics of the allosteric interactions of phosphofructokinase from Escherichia coli

Abstract Phosphofructokinase has been partially purified from Escherichia coli , and its kinetic properties investigated. It shows co-operative interactions with respect to one of its substrates, fructose-6-phosphate, but not towards the second, namely ATP. ADP and other diphosphonucleosides act as activators and phosphoenolpyruvate as an inhibitor. Both effectors decrease the homotropic interactions between fructose-6-phosphate molecules; but, whereas the activators increase the affinity of the enzyme for this substrate, the inhibitor decreases it. These ligands have no effect on the maximum velocity of the reaction, except in the case of ADP which is a competitive inhibitor of ATP. These homotropic and heterotropic interactions are qualitatively and quantitatively accounted for by the concerted transition theory proposed by Monod, Wyman & Changeux (1965), assuming the enzyme to be in equilibrium between two conformational states which differ in their dissociation constants for fructose-6-phosphate, activators and inhibitor. A convenient method of obtaining these intrinsic dissociation constants has been derived from the equations of the theory. From the kinetic data, it is also possible to obtain the value of the equilibrium constant between the two states, if it is assumed that the enzyme is a tetramer made up of four identical subunits and that the transition is perfectly concerted.

Studies on the induced synthesis of β-galactosidase in Escherichia coli: The kinetics and mechanism of sulfur incorporation

Abstract A study of the kinetics of sulfur incorporation into the molecule of β-galactosidase during the induced synthesis of this enzyme in E. coli brings proof that the enzyme-protein is synthesized entirely de novo without any appreciable participation of materials coming from other cellular proteins. Furthermore, there is no measurable renewal of β-galactosidase sulfur in growing cells whether or not the enzyme is being synthesized. The induced synthesis of β-galactosidase appears as a virtually irreversible process. The bulk of the other cellular proteins in E. coli are equally stable and do not undergo any appreciable degradation and resynthesis during growth. The apparent contradiction between these results and the generally accepted concepts regarding the dynamic state of intracellular proteins is discussed.

Purification et propriétés de la β-galactosidase (lactase) d'escherichia coli☆

1. 1. A method for the purification of the adaptive enzyme of E. coli, β-galactosidase (lactase) has been described. 2. 2. Lactose and o-nitrophenyl-β-d-galactopyranoside are quantitatively hydrolysed by this enzyme preparation which, however, is inactive on sugars not possessing the β-galactosidic configuration. 3. 3. The following results (4, 5, and 6) indicate that the hydrolysis of lactose and o-nitrophenyl-β-d-galactopyranoside i is catalyzed by the same enzyme. 4. 4. The speed of thermal inactivation is the same whether one determines the activity with lactose or o-nitrophenyl-β-d-galactopyranoside as substrate. 5. 5. The electrophoretic analysis of this purified preparation reveals the presence of at least two constituents. Each of these electrophoretically distinct constituents is active on lactose and on o-nitrophenyl-β-d-galactopyranoside, and the ratio of these activities is the same for both fractions. 6. 6. A precipitating anti-lactase serum has been obtained by immunization of rabbits with the purified enzyme preparation. A quantitative study of the precipitin reaction indicates that the enzymatic activity, whether it be determined on lactose or o-nitrophenyl-β-d-galactopyranoside, is associated with a single antigenic species. There has also been demonstrated the existence of a protein which possesses no enzymatic activity but which markedly crossreacts with the β-galactosidase. 7. 7. The action of various ions on the enzymatic hydrolysis of lactose has been investigated. The effects of monovalent cations are particularly marked and complex. Depending upon the conditions (the presence of certain other cations), a given ion (sodium) is able to behave as either an inhibitor or an activator. 8. 8. These effects are the result of the competetive relationship between the monovalent cations. In order to account for the above phenomena, a hypothesis is presented, bringing into play only two characteristic constants: the affinity (inverse of the apparent dissociation-constant of ion-enzyme complex) and the “activance” (activity of the enzyme when saturated by the ion). 9. 9. For the hydrolysis of lactose, potassium shows the maximum “activance”, while the sodium is only weakly active. On the other hand, with o-nitrophenol-β-d-galactopyranoside as substrate, the “activance” of sodium is higher than that of the potassium. This remarkable inversion of the effects of ions demonstrates that the activance is linked to the chemical structure of the substrate.

NON-INDUCIBLE MUTANTS OF THE REGULATOR GENE IN THE "LACTOSE" SYSTEM OF ESCHERICHIA COLI.

Two independent mutants of Escherichia coli K12 have been isolated which have lost the capacity to be induced by β -galactosides for the production of the proteins governed by the lactose Operon in E. coli . These mutations are located in the regulator ( i ) gene of the Lac region. In heterogenotes, the mutated i s allele is dominant over the wild i + allele. Lac + revertants are obtained as a result of a secondary mutation either in the i gene ( i − ) or in the operator ( o c ). The i s mutations appear to result in an alteration of the lactose repressor which becomes unable to respond to β -galactosides.

Rôle du lactose et de ses produits métaboliques dans l'induction de l'opéron lactose chez Escherichia coli

Abstract Role of lactose and its metabolic products in the induction of the lactose operon in Escherichia coli It is shown that lactose is not an inducer of the “Lac” operon in Escherichia coli K-12. Induction of this operon in the presence of lactose results from transgalactosidation reactions for which β-galactosidase (EC 3.2.1.23) itself is responsible. Several compounds obtained by the action of galactosidase on a mixture of lactose and various alcohols have been isolated and shown to be inducers of the system.

On the subunit structure of wild-type versus complemented β-galactosidase of Escherichia coli☆

Abstract Various properties of wild-type galactosidase from Escherichia coli were compared with those exhibited by enzyme extracted from complementary diploids. It is concluded that whereas the complemented enzyme is made by the aggregation of peptide fragments derived from each of the two structural genes present in the diploid, each protomer of the wild-type enzyme behaves as a single covalent unit. The z gene must therefore be regarded as consisting of a single cistron. The mechanism of this apparently new type of intracistronic complementation is briefly discussed.

On the expression of a structural gene

Experiments were made on the kinetics of β -galactosidase production by zygotes formed upon mating of inducible, lac + (Hfr z + i + ), and constitutive, lac − (F − z − i − ), strains of Escherichia coli K12. Enzyme formation commenced within two minutes of the time of injection of the z + gene. The zygote thereafter produced β -galactosidase at a rate similar to that by an induced bacterium, and no increase in rate of enzyme production per zygote was observed in the first thirty minutes after mating. Therefore, the time required for the injected genetic material to become active in the functional apparatus of the F − cell, for formation of any possible intermediates between genetic material and enzyme, and for formation of the enzyme itself, are each less than two minutes. The Hfr strain of E. coli was labeled with 32 P and was mated with the non-radioactive F − bacteria. A segment of 32 P-labeled genetic material containing the z + locus was transferred to the recipient. 32 P decay in the newly acquired genetic material was found to decrease the enzyme-forming capacity of zygotes even when it occurred some time after initiation of enzyme synthesis. These results appear to indicate that integrity of the genetic material is required not only for an initial transfer of genetic information, but actually for enzyme synthesis to continue.

Délétions fusionnant ľopéron lactose et un opéron purine chez Escherichia coli

From a strain of Escherichia coli K12, which carries an episome F′ and is thereby diploid for the region Lac-Pro2-Ph-T6-Pur, a series of 68 mutants has been isolated in which a fragment of the material carried by the episome is deleted. On one side, all these deletions extend to various sites of gene z. They all cover o, i, Pro2 and Ph. On the other side, 2 of them extend to the region between Ph and T6, 50 extend to the region between T6 and Pur, and 16 extend into one cistron (β), while respecting another cistron (α), of the Pur region. In the 16 deletions which extend into Pur β, the synthesis of the β-galactoside permease and transaeetylase is no longer induced by β-galactosides but repressed by addition of purine in the medium. It appears that in these 16 cases, the deletions have joined a part of the Lac operon to a part of a Pur operon, the new operon which is thus formed being submitted to a repressive regulation by purines.