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H Buc

Publications and source records attributed to H Buc.

At least 91 records · Page 5Linked to original sources
PubMed1982

A transcriptionally active plasmid-protein complex isolated from Escherichia coli.

A stable transcriptionally active plasmid-protein complex has been isolated in high yield from Escherichia coli containing the thermally-inducible plasmid pKN 402A. The complexes which have a protein/DNA weight ratio of approx. 1 contain more than 11 polypeptide species. The weight percents of the three known proteins in the complex H1, RNA polymerase and HU, are 23, 23 and 5%, respectively. In vitro RNA synthesis by this complex proceeds for several hours and is inhibited by rifampicin and actinomycin to 33 and 98%, respectively, suggesting that most of the observed nucleotide incorporation is due to elongation of preinitiated RNA chains. Exogenous E. coli RNA polymerase but not exogenous DNA stimulates the in vitro transcription indicating that RNA polymerase is limiting and binds tightly to the plasmid. Stimulation of the in vitro transcription by the addition of exogenous E. coli core polymerase suggests that sigma subunit may be released in the RNA synthesis. This transcriptionally active complex should prove to be useful to study the mechanism of transcription and regulation in vivo.

Bacterial Proteins↗
PubMed1982

Is DNA unwound by the cyclic AMP receptor protein?

Superhelical pBR 322 derivatives have been relaxed by eukaryotic topoisomerase I in the presence or in the absence of E. coli cyclic AMP receptor protein (CRP) and of cyclic AMP (cAMP). CRP alone, or cAMP alone do not affect the average linking number of the distribution of the relaxed topoisomers. Hence, they do not unwind the template. In the presence of cAMP, CRP induces a small unwinding. The extent of this unwinding is barely modified when the relaxation is carried out on a similar vector plasmid where the CRP binding site of the lac or of the gal operon has been inserted. Under these conditions, we checked that CRP occupies the lactose control site and that upon addition of RNA polymerase, the corresponding promoter is readily activated. These findings are difficult to reconcile with the proposal that activation of these promoters results from the binding of the CRP-cAMP complex to left-handed DNA sequences.

Animals↗
PubMed1982

Point mutations change the thermal denaturation profile of a short DNA fragment containing the lactose control elements. Comparison between experiment and theory.

To understand the denaturation process of short DNA segments we have chosen a 203-base pair (bp) restriction fragment containing the lactose control region. A steady decrease in GC content exists between its i proximal and z proximal ends. We confirm that this fragment melts at low salt in two subtransitions. A GC to AT mutation in the AT-rich region (mutation UV5) increases the number of denatured base pairs in the first subtransition and decreases the cooperativity of the melting process. A GC to AT mutation in the GC-rich region (mutation L8) decreases the number of denatured base pairs in the first subtransition and increases the cooperativity. These mutations induce the same shift in the temperature of half denaturation. The effects of both mutations are additive. A short deletion at the z end of the fragment affects only the first subtransition. When four GC pairs are added to both end, the fragment melts in one transition. Comparison with the results obtained with a larger 789-bp lac fragment reveals strong end effects on base pair stability and suggests that denaturation of the 203-bp fragment proceeds unidirectionally from the z end. Good agreement is shown with the predictions made with the "z ipper model" of Crothers et al. (1965).

Base Sequence↗
PubMed1981

On the binding of tRNA to Escherichia coli RNA polymerase. Interactions between the core enzyme, DNA and tRNA.

We have investigated the interplay between the binding of tRNA and DNA to core RNA polymerase. We show that the monomer core enzyme can bind stably to either DNA or tRNA, whereas the dimer core can fix both DNA and tRNA in a stable ternary complex. We have examined the kinetics of the exchange between DNA and tRNA bound to the core enzyme. DNA bound to monomer core can be rapidly displaced by tRNA without prior dissociation of the core from the DNA. Similarly tRNA bound to the core can be displaced by DNA without prior dissociation of the tRNA. We confirm the result of Hinkle and Chamberlin [J. Mol. Biol. 70, 157-185 (1972)] that, in contrast, the core enzyme must first dissociate from one DNA molecule before it can transfer to another DNA. As this dissociation is very slow we suggest that, in vivo, the tRNA can act as a 'porter' providing the core enzyme with a more kinetically favourable path to transfer from one DNA site to another.

Bacterial Proteins↗
PubMed1981

Mechanism of allosteric activation of glycogen phosphorylase probed by the reactivity of essential arginine residues. Identification of an arginine residue involved in the binding of glucose 1-phosphate.

We have previously reported the physicochemical and kinetic properties of glycogen phosphorylase modified by arginine-specific reagent under different conditions [Dreyfus, M., Vandenbunder, B., & Buc, H. (1980) Biochemistry 19, 3634-3642]. The properties of the modified enzyme depend upon the conformation adopted by the enzyme during the modification reaction. In this paper, we report the localization of the crucial modified arginine residues on the primary structure. The chymotryptic peptide extending from residue Asp-563 to residue Tyr-572 was shown to contain one arginine residue (Arg-568) which is chemically modified by phenylglyoxal in phosphorylase a and in activated phosphorlase b. Inclusion of glucose 1-phosphate in the modification medium protects this residue from modification, with a concomitant protection of the enzyme activity. Furthermore, this residue is not reactive toward phenylglyoxal in phosphorylase b in the absence of any effector. Addition of the AMP analogue 2'dAMP, which is not an activator of the enzyme, does not increase Arg-568 reactivity but protects from modification several arginine residues located between Arg-242 and Leu-348. The location and the role of Arg-568 in phosphorylase are discussed with reference to recent data from X-ray crystallography.

Allosteric Regulation↗
PubMed1981

Regulatory properties of phosphofructokinase 2 from Escherichia coli.

Escherichia coli K12 contains two phosphofructokinases: phosphofructokinase 1, the most studied one, appears to behave as an allosteric enzyme, while phosphofructokinase 2 presents the features of a Michaelian enzyme. We show the present paper that, in fact, phosphofructokinase 2 also presents some regulatory properties in vitro: at high concentrations, ATP is an inhibitor of phosphofructokinase 2 and it provokes the tetramerization of the dimeric native enzyme. The binding of the two substrates to phosphofructokinase 2 is sequential and ordered as for phosphofructokinase 1, but in the former case fructose 6-phosphate is the first substrate to be bound and ADP the first product to be released. Each dimer of phosphofructokinase 2 binds two molecules of fructose 6-phosphate but only one molecule of the product fructose 1,6-phosphate. Although both phosphofructokinases of E. coli K12 present regulatory properties in vitro, the mechanism of regulation of the activity of the two enzymes is strikingly different. It can be asked whether or not these mechanisms operate in vivo.

Escherichia coli↗
PubMed1980

Prokaryotic histone-like protein interacting with RNA polymerase.

firA mutation of Escherichia coli can render RNA synthesis thermosensitive and confer abnormal sensitivity to rifampicin, an antibiotic that specifically inhibits the activity of RNA polymerase. We previously described the cloning of a chromosomal HindIII fragment containing the firA gene, and we now present strong evidence that the product of this gene is a 17,000-dalton polypeptide which, by various criteria, closely resembles the eukaryotic histones. This protein forms the largest of a unique set of three abundant histone-like proteins (HLP) found in E. coli and is hence referred to as HLPI. We discuss possible routes by which these proteins might affect transcription.

Bacteriophage lambda↗
PubMed1980

Phosphorylation of human erythrocyte pyruvate kinase by soluble cyclic-AMP-dependent protein kinases. Comparison with human liver L-type enzyme.

Human red cell contain soluble adenosine-3',5'-phosphate-dependent protein kinases, which are able to phosphorylate the L' subunits of erythrocyte pyruvate kinase. Efficiency and maximum level of phosphorylation are very comparable in human liver and red cells. Phosphorylation of red cell pyruvate kinase results in the same kinetic modifications as for liver enzyme, namely a shift towards a 'T' allosteric state characterized by a decreased affinity for phosphoenolpyruvate and increased inhibition by the allosteric inhibitors ATP and alanine. In the course of red cell aging a small amount of partially proteolysed pyruvate kinase, devoid of the phosphorylatable site, appears; it resembles the subtilisin-treated L'4 enzyme and accounts for less than 20% of total pyruvate kinase subunits. Endogenous phosphorylation of pyruvate kinase from erythrocytes incubated in the presence of cyclic nucleotides produces the same kinetic modifications as phosphorylation in partially purified extract; this, however, does not change glucose consumption, lactate production and glycolytic intermediate concentrations of the incubated cells.

Cyclic AMP↗
PubMed1979

[Chemical reactivity of an essential arginine residue in substrate binding, reflecting the state of activation of glycogen phosphorylase in rabbit muscle].

Arginine residues have been shown to be present in the binding sites of both phosphorylated substrates and nucleotide activators of glycogen phosphorylase. The former residue can only be modified by arginine-directed reagents when the enzyme is activated, i. e. in phosphorylase a, or in phosphorylase b in the presence of activators. Conversely, after this residue has been modified, phosphorylase b is irreversibly blocked in an active conformation.

Adenosine Monophosphate↗
PubMed1979

Isolation of plasmid-protein complexes from Escherichia coli.

A procedure is described for the isolation of complexes between pMB9 plasmids and protein from Escherichia coli which are stable during centrifugation on sucrose gradients and are not destroyed in the presence of competitor DNA. The proteins in these complexes have been analysed by dodecyl sulphate/polyacrylamide gel electrophoresis. Only 10 polypeptide species are found in significant quantities, many of which are bound to both the plasmid and host DNA. We have also detected the presence of one protein which binds to a specific DNA sequence inserted in the plasmid.

Bacterial Proteins↗
PubMed1979

On the binding of tRNA to Escherichia coli RNA polymerase.

The fixation of tRNA to Escherichia coli RNA polymerase has been investigated. Bound and free tRNA have been separated and quantified after filtration through cellulose nitrate filters, centrifugation or sucrose gradients or electrophoresis in polyacrylamide gels. We detect no differences between the fixation of E. coli fMet-tRNAfMet, Met-tRNAmMet or uncharged unfractionated tRNA to RNA polymerase. Tight complexes, with a long residence time, are formed between core enzyme and tRNA with a dissociation constant of less than 1 nM. Complexes exist between tRNA and both monomer and dimer forms of the core enzyme. In the monomer complex, one tRNA is bound per alpha 2 beta beta' unit, whereas in the dimer complex only 0.5 tRNA molecule is fixed per alpha 2 beta beta' unit. In contrast to the core enzyme, very little tRNA fixes tightly to the holoenzyme at salt concentrations greater than 80 mM. At lower salt concentrations tRNA fixation results in a loss of sigma subunit from the holo enzyme to the resulting core enzyme where it binds tightly. DNA fixation reduces the binding of tRNA to RNA polymerase and tRNA fixation reduces the binding of DNA. However, binding of DNA to polymerase is not competitive with binding of tRNA, and ternary complexes between RNA polymerase, DNA and tRNA are shown to exist. Our results are discussed in relation to other studies concerning the effects of tRNA upon RNA polymerase.

DNA↗
PubMed1979

The interplay between covalent and non-covalent regulation of glycogen phosphorylase. The role of different effectors of phosphorylase b on the phosphorylase b to a conversion rate.

Glycogen phosphorylase b is converted to glycogen phosphorylase a, the covalently activated form of the enzyme, by phosphorylase kinase. Glc-6-P, which is an allosteric inhibitor of phosphorylase b, and glycogen, which is a substrate of this enzyme, are already known to have respectively an inhibiting and activating effect upon the rate of conversion from phosphorylase b to phosphorylase a by phosphorylase kinase. In the former case, this effect is due to the binding of glucose-6-phosphate to glycogen phosphorylase b. In order to investigate whether or not the rate of conversion of glycogen phosphorylase b to phosphorylase a depends on the conformational state of the b substrate, we have tested the action of the most specific effectors of glycogen phosphorylase b activity upon the rate of conversion from phosphorylase b to phosphorylase a at 0 degrees C and 22 degrees C : AMP and other strong activators, IMP and weak activators, Glc-6-P, glycogen. Glc-1-P and phosphate. AMP and strong activators have a very important inhibitory effect at low temperature, but not at room temperature, whereas the weak activators have always a very weak, if even existing, inhibitory effect at both temperatures. We confirmed the very strong inhibiting effect of Glc-6-P at both temperatures, and the strong activating effect of glycogen. We have shown that phosphate has a very strong inhibitory effect, whereas Glc-1-P has an activating effect only at room temperature and at non-physiological concentrations. The concomitant effects of substrates and nucleotides have also been studied. The observed effects of all these ligands may be either direct ones on phosphorylase kinase, or indirect ones, the ligand modifying the conformation of phosphorylase b and its interaction with phosphorylase kinase. Since we have no control experiments with a peptidic fragment of phosphorylase b, the interpretation of our results remains putative. However, the differential effects observed with different nucleotides are in agreement with the simple conformational scheme proposed earlier. Therefore, it is suggested that phosphorylase kinase recognizes differently the different conformations of glycogen phosphorylase b. In agreement with such an explanation, it is shown that the inhibiting effect of AMP is mediated by a slow isomerisation which has been previously ascribed to a quaternary conformational change of glycogen phosphorylase b. The results presented here (in particular, the important effect of glycogen and phosphate) are also discussed in correlation with the physiological role of the different ligands as regulatory signals in the in vivo situation where phosphorylase is inserted into the glycogen particle.

Adenosine Monophosphate↗
PubMed1978

A radioassay for pyrimidine-5'-nucleotidase activity.

A simple method for the determination of pyrimidine-5'-nucleotidase activity in red cells is described. The radioactive uridine released after incubation with [5-3H]] uridine 5-monophosphate (UMP) is separated on DEAE-cellulose paper and counted. This method does not require preliminary dialysis of the hemolysate and is 50-fold more sensitive than that based on the measurement of the inorganic phosphate released. One patient with pyrimidine-5'-nucleotidase deficiency was detected with this method.

Erythrocytes↗