François Chapeville

Purification and properties of Escherichia coli CTP(ATP)-tRNA nucleotidyltransferase

Abstract 1. CTP(ATP)-tRNA nucleotidyltransferase has been purified 1000-fold from Escherichia coli. It appears homogeneous on analytical centrifugation and about 60% pure on disc electrophoresis. 2. The enzyme requires Mg2+ or Mn2+. 3. The molecular weight of the enzyme is 37000 and the sedimentation coefficient 2.9 S. 4. The Michaelis constants are 3.3 · 10−4 M and 1.7 · 10−5 M for ATP and CTP, respectively. 5. ATP is a non-competitive inhibitor of CMP incorporation into tRNA and CTP a non-competitive inhibitor of AMP incorporation. Ki for ATP is 3.7 · 10−4 M and for CTP 2.0 · 10−4 M.

Qualitative and quantitative distribution of plasminogen activators in organs from healthy adult mice

Twenty organs from healthy adult mice were tested for plasminogen activator activity. All were positive although specific activities varied 200‐fold. Tissues with high activity were lung, uterus, brain and kidney. Endocrine glands were moderately rich in activator activity, and lymphoid tissues were poor. Molecular mass characterization was carried out. Two enzymatic forms were observed in all twenty organs: a 70 kDa form similar to human tissue plasminogen activator and a 48 kDa form analogous to mouse urokinase.

Chick embryo poly (rA:dT)-dependent DNA polymerase

It has been shown in several laboratories that RNA oncogenic viruses contain an RNA-dependent DNA polymerase activity (reverse transcriptase) [ 1, 21 associated with DNA-dependent polymerase and DNA ligase activities [3,4]. According to Temin’s DNA provirus hypothesis [S] this system may be involved in the replication of the RNA tumor viruses through a DNA intermediate. Similar enzymatic activities are found also in transformed mammalian cells [6, 71 and even in normal cells [7, 81 . RNA-dependent DNA polymerases from virions and transformed cells can use as template doublestranded homopolymers of the type (rA:rU) or (rA:dT) [9]. On the other hand, the enzyme from normal cells uses preferentially (rA:dT) polymer [7, 81. This fact and other more recent results suggest that the enzymes prepared from these two kinds of materials are different. The hypothetical role of these enzymes in gene amplification during cell differentiation and the possibility that the virion enzyme might be of cellular origin led us to investigate whether reverse transcriptase activity is also present in embryonic tissues. A poly (rA:dT)-dependent DNA polymerase has been characterized and purified about 1000 times from the heart of chick embryos. This enzyme specifically copies the poly A strand of the (rA: dT) hybrid and very weakly poly A alone. The enzyme is distinct from at least two DNA-dependent DNA polymerases also present in tissue extracts.

Chemical Recognition in Biology

A. Recognition of Ligands - Enzymic Catalysis.- 1. What Everyone Wanted to Know About Tight Binding and Enzyme Catalysis, but Never Thought of Asking.- 2. The Cytochromes c: Paradigms for Chemical Recognition.- 3. Recognition of Ligands by Haem Proteins.- 4. Influences of Solvent Water on the Transition State Affinity of Enzymes, Protein Folding, and the Composition of the Genetic Code.- 5. Suicide Substrates: Mechanism-Based Inactivators of Specific Target Enzymes.- 6. Recognition: the Kinetic Concepts.- 7. Coupled Oscillator Theory of Enzyme Action.- 8. Stereochemical Aspects of Chain Lengthening and Cyclization Processes in Terpenoid Biosynthesis.- B. Enzyme Regulation.- 1. Three Multifunctional Protein Kinase Systems in Transmembrane Control.- 2. Effect of Catabolite Repression on Chemotaxis in Salmonella typhimurium.- 3. Subunit Interaction of Adenylylated Glutamine Synthetase.- 4. Dynamic Compartmentation.- 5. The Genes for and Regulation of the Enzyme Activities of two Multifunctional Proteins Required for the De Novo Pathway for UMP Biosynthesis in Mammals.- 6. Regulation of Muscle Contraction by Ca Ion.- 7. Why is Phosphate so Useful?.- 8. ppGpp, a Signal Molecule.- 9. Gramicidin S-Synthetase: On the Structure of a Polyenzyme Template in Polypeptide Synthesis.- 10. A Molecular Approach to Immunity and Pathogenicity in an Insect-Bacterial System.- C. Nucleic Acid - Protein Interactions Mutagenesis.- 1. Structure of the Gene 5 DNA Binding Protein from Bacteriophage fd and its DNA Binding Cleft.- 2. Recognition of Nucleic Acids and Chemically-Damaged DNA by Peptides and Proteins.- 3. Specific Interaction of Base-Specific Nucleases with Nucleosides and Nucleotides.- 4. Structural and Dynamic Aspects of Recognition Between tRNAs and Aminoacyl-tRNA Synthetases.- 5. Recognition of Promoter Sequences by RNA Polymerases from Different Sources.- 6. DNA as a Target for a Protein Antibiotic: Molecular Basis of Action.- 7. Site-Specific Mutagenesis in the Analysis of a Viral Replicon.- D. Protein Biosynthesis.- 1. Molecular Mechanism of Protein Biosynthesis and an Approach to the Mechanism of Energy Transduction.- 2. On Codon - Anticodon Interactions.- 3. Fluorescent tRNA Derivatives and Ribosome Recognition.- 4. Structure and Evolution of Ribosomes.- E. Philosophical Reflexions.- 1. Molecular Biology, Culture, and Society.- 2. Personal Recollections of Fritz Lipmann During the Early Years of Coenzyme A Research.

Peptidyl-tRNA hydrolase: Mise en évidence dans différents organismes. Activité enzymatique en présence des ribosomes

Summary Already described in bacteria, the peptidyl-tRNA hydrolase, which catalyzes the hydrolysis of the ester linkage that binds an N-acyl-amino acid or a polypeptide to tRNA, is shown here to be also present in plant and animal cells. In E. coli , 20 p. 100 of the enzyme is recovered bound to ribosomes, and the remaining 80 p. 100 is free. The ribosome-bound enzyme retains its activity. The enzyme can be removed from the ribosomes by repeated washing with 0.1 M ammonium chloride in normal undissociating conditions and also in the presence of low magnesium ion concentrations which dissociate the 70 S particles into their subunits. Magnesium ions appear to be involved in the binding of the enzyme to the ribosomes. When the enzyme is incubated in the presence of the acPhe-tRNA—ribosome—polyU complex, it does not hydrolyze the bound acPhe-tRNA.

Plant viruses and new perspectives in cross-protection

Cross-protection in plants is the phenomenon whereby a plant preinoculated with a mild virus strain becomes resistant to subsequent inoculation by a related severe strain. It has been used on a large scale in cases where no resistant plants are available. Although several hypotheses have been proposed to explain the molecular mechanism underlying cross-protection, no single hypothesis can account for all the data obtained. Recently, a phenomenon akin to cross-protection has been achieved in transformed plants harboring the cDNA of a part of a viral RNA genome. These results obtained by genetic engineering raise new hopes for obtaining plants resistant to virus infection.

On the mechanism of nucleotide incorporation into DNA and RNA

The discovery of the exonuclease activity associated to some DNA polymerases [l-3] gave support to the notion that accuracy of polymerization with these enzymes is related to the kinetic interlocking of the exonuclease to the polymerase activities [2,3] although no satisfactory model is available to date. Eukaryotic DNA polymerases have no associated exonuclease activity [4] and yet may replicate templates with high fidelity [ 51. Our studies on the dynamics of recognition processes [6-81 led us to the suggestion of a new possibility: kinetic amplification of discrimination [8] . Very similar conclusions were independently reached by J. Hopfield [9]. Thus, reaction schemes were described which allowed a much better discrimination between correct and incorrect substrate than would be predicted from the simple comparison of the kinetic parameters of the reaction carried by an enzyme on the two substrates [8,9]. We have performed kinetic experiments with DNA polymerase I and RNA polymerase from E. coli. Both enzymes are able to convert nucleoside triphosphates into nucleoside diphosphates in the course of the polymerization reaction on synthetic templates. The qualitative and quantitative aspects of the phenomenon are suggestive of a mechanism of incorporation using kinetic proofreading in Hopfield’s sense [9].

Developmental changes in terminal deoxynucleotidyl transferase of the chicken thymus.

Abstract Terminal deoxynucleotidyl transferase activity begins to be detectable in the thymus of 14-day old chicken embryos. It reaches a maximum 3 weeks after hatching, and persits at a fairly high level in 21-weeks old chickens. Multiple chromatographic forms of TdT are detected, and the relative proportions of these forms change during the development of the chicken.

Study of the Escherichia coli tRNA nucleotidyltransferase: Effect of inorganic ions and thiol blocking reagents on enzyme activity

Abstract The effect of different divalent and monovalent cations on reactions catalyzed by the Escherichia coli tRNA nucleotidyltransferase has been examined. The highest rates of incorporation of AMP into tRNA—pX—C—C or tRNA—pX and of CMP into tRNA—pX are observed in the presence of Mg2+. Mn2+ is less efficient than Mg2+ for AMP incorporation; it is inefficient for CMP incorporation; in its presence UMP is incorporated instead of CMP. Co2+ shows only low efficiency in all cases examined. In the presence of optimal Mg2+ concentration, Mn2+ decreases the rate of incorporation of CMP and, to a lower extent, of AMP, but increases the rate of UMP incorporation. K+ at 0.1 M concentration stimulates CMP incorporation in both tRNA—pX and tRNA—pX—C whereas it has an inhibitory effect on AMP and UMP incorporation in all cases. The role of thiol groups on enzyme activity has been examined using —SH blocking reagents. 5,5′-Dithiobis(2-nitrobenzoic) acid or N-ethylmaleimide inhibit the incorporation of AMP into tRNA—pX—C—C but are without effect on the incorporation of CMP or UMP into tRNA—pX.

Study of the Escherichia coli tRNA nucleotidyltransferase. Interactions of the enzyme with tRNA.

Abstract A complex between Escherichia coli tRNA nucleotidyltransferase and tRNA has been isolated by centrifugation in a glycerol gradient (sedimentation constant 5.3 S). The complex is dissociated in the presence of ethylenediamine tetraacetate or high salt concentrations. The apparent K m of the enzyme for E. coli tRNA—pX—C—C in the reaction of incorporation of AMP is 1.5 · 10 −6 M; tRNA with an intact 3′ terminus is a competitive inhibitor of the tRNA substrate in this reaction, with a K i value of 0.9 · 10 −6 M. tRNA protects tRNA nucleotidyltransferase against thermal inactivation. The activation energy of the reaction of inactivation of tRNA-complexed tRNA nucleotidyltransferase is twice that of the free enzyme. The dissociation constant of the tRNA—tRNA nucleotidyltransferase complex deduced from the protection is 0.3 · 10 −6 M for tRNA—pX—C—C—A. Intercalation of ethidium bromide into tRNA molecules reduces the rate of the reaction catalyzed by tRNA nucleotidyltransferase but not the extent of the addition of nucleotides; the addition of this dye to the tRNA phosphate groups decreases both. Besides the synthesis of the 3′ terminal —pC—C—A sequence of tRNA molecules and of certain viral RNAs, the tRNA nucleotidyltransferase exhibits a low activity of incorporation of CMP into ribosomal RNA partially degraded by phosphodiesterase.