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M A Jacquet

Publications and source records attributed to M A Jacquet.

9 recordsLinked to original sources
PubMed1999

The kinetics of sigma subunit directed promoter recognition by E. coli RNA polymerase.

Time-resolved laser UV irradiation and controlled proteolysis have been used to study the sequential recognition of the lac UV5 promoter by Escherichia coli RNA polymerase. Local rearrangements in the DNA, the appearance of intimate protein-DNA contacts, and structural changes within the sigma subunit together provide specific signatures that define major species populated during this process. At 22 degreesC, a first closed complex is characterised by a transient conformational change in the sigma subunit and by a distortion in the -35 region. Subsequently, direct contacts at -34 and at positions -8, -5 and -3 on the non-template strand appear prior to DNA strand separation. The contact in the -35 consensus region involves only the sigma subunit. This intermediate possesses different structural parameters from that formed by quenching open complexes from 37 degreesC to 14 degreesC. Sigma thus appears as the principal partner acting during promoter recognition, a strongly coupled process involving two major intermediates only.

DNA, Bacterial↗
PubMed1992

In vivo control of promoter and terminator efficiencies at a distance.

In pBR329, the genes providing resistance to ampicillin (beta-lactamase, bla) and chloramphenicol (chloramphenicol acetyl transferase, cat) are encoded on the same strand. The bla gene lies downstream of the cat gene, separated by an intergenic sequence of 414 bp. The transcription starts of the two genes are 1090 bp apart. We have probed, in vivo, the effect on transcription of the bla gene, of the introduction, in front of the cat gene, of a series of synthetic promoters covering a large (over 60-fold) range of efficiency. The rising efficiency of the cat promoter has several important consequences for transcription of the bla gene. First, a strong (up to sevenfold) stimulation of the bla promoter is observed, together with a shift of the main bla transcription start site, 10 bp upstream. Furthermore, the relative efficiencies of the bla transcription terminators are reduced. Finally, because of a lesser relative efficiency of the cat transcription terminators as well, we observe enhanced intrusion into the bla gene of transcripts initiated at the cat promoter, some of them extending to the bla transcription terminator and beyond. The operon-like expression of the cat-bla gene tandem is controlled by the efficiency of the cat terminator, which in turn depends on that of the cat promoter. This demonstrates a direct link between the efficiencies of promoter and terminator. Upon inhibition of bacterial gyrase activity, i.e. relaxation of negative supercoiling action, bla expression increases sharply in pBR329, but remains almost unchanged in a plasmid (pBRGC-1) in which cat is under the control of a 6.5-fold stronger promoter. Therefore, under normal gyrase activity, the stimulation of the bla promoter in pBRGC-1 (relative to pBR329) appears to be linked to topological relaxation of its template in situ, in keeping with earlier in vitro observations. We propose that the relaxed state of pBRGC-1 in situ could be due to the decrease in the plasmid linking number, introduced by the 10-12 RNA polymerases that simultaneously transcribe the cat gene in that plasmid, compared with only one or two in pBR329. We find that the negative superhelical densities of both plasmids are almost identical when extracted from the cell. Therefore gyrase would not correct for the relaxed state of plasmid pBRGC-1 observed in situ.

Ampicillin Resistance↗
PubMed1990

Transcription in vivo directed by consensus sequences of E.coli promoters: their context heavily affects efficiencies and start sites.

We studied in vivo transcription and gene expression directed by a series of synthetic sequences, bearing the consensus hexamer (CH) pair of E.coli promoters in various contexts. The results demonstrate that, for the contexts tested, the CH pair supports transcription activity and gene expression, whether the spacer linking them is AT or GC rich, or is as short as 14 bp or as large as 26 bp (standard size 17 bp). However, we find that the context influences transcription efficiency by as much as an order of magnitude, and is able to scatter transcription start sites over a region of as much as 30 bp, including start sites within a CH or even between the two sequences of the CH pair. The results demonstrate that, although the CH pair can be sufficient for directing transcription by E.coli RNAP, important determinants for promoter activity are at least in part contained in the context of the consensus sequences; they advocate a synergic interplay of signals borne by the CH pair and its context, extending over all parts of the promoter sequence. A two-step model is proposed, in which properly located consensus sequences provide RNAP with facilities required for stereospecific docking along the promoter sequence; the result would be a sharp change in the local environment of the double helix inducing local isothermal unwinding. The size of the loop (related to the AT constraint in the promoter) and the extend of the environmental change required for unwinding would determine the rate of transcriptionally competent complex formation, positioning and grouping of start sites.

Base Sequence↗
PubMed1989

In vivo gene expression directed by synthetic promoter constructions restricted to the -10 and -35 consensus hexamers of E. coli.

Two synthetic DNA sequences, carrying no other known E. coli promoter element than the consensus hexamers (CH) TTGACA (CH-35) and TATAAT CH(-10), spaced by 17 bp, were inserted in pBR329, in a position enabling transcription of the complete Cmr gene. The region upstream of the Cmr transcription start was carefully cleared of w.t. promoter elements (full deletion of the wild type (w.t.) Cmr promoter upstream +2 and large portion of an upstream coding sequence). Both synthetic promoters, which differ only by the sequences of the spacers (non consensus, constrained in AT or GC) support in vivo high level Cmr gene expression. The GC rich spacer is associated with transcription start at the usual +1 position, but with the AT rich spacer, transcription starts at several places, mainly in CH(-10). Rearranged promoter sequences derived from the synthetic ones upon transformation with partly ligated plasmids, yield new insights on the role of the standard CH pair, the size of the spacer and the sequence downstream of CH(-10).

Base Sequence↗
PubMed1985

In vitro transcription initiation from three different Escherichia coli promoters. Effect of supercoiling.

Transcription initiation from beta-lactamase, tetracycline resistance and RNA 1 promoters, present in plasmid pAT153, were studied employing the abortive initiation technique. Assays appear to be promoter-specific with supercoiled and linear templates. Supercoiling enhances the isomerization rate constant of the open RNA-polymerase--promoter complex formation. Results agree with the in vivo behaviour of the corresponding promoters, and allow us to propose a hypothesis about the effect of supercoiling on transcription initiation.

DNA, Superhelical↗
PubMed1985

In vivo and in vitro effect of mutations in tetA promoter from pSC101: insertion of poly(dA.dT) stretch in the spacer region does not inactivate the promoter.

Two mutants, mapping at the HindIII site (between the consensus sequences) of the pSC101 tetA promoter, were studied: MA2 corresponds to a 4 bp deletion between positions -12 and -15; B30 bears a 44 bp insertion C(TA)21 G at the HindIII site. Both mutants were assayed in vivo (ability of the plasmid to confer resistance to tetracycline, plasmid-directed protein synthesis, S1-mapping of mRNA) and in vitro (abortive initiation assay). Compared to w.t., MA2 is a poor promoter in vivo; RNA polymerase binding, complex activation and rate of initial oligonucleotide synthesis are strongly reduced in vitro; this is in keeping with the known effects of altering the consensus elements in E. coli promoters. In contrast, B30 shows in vivo a promoter activity only slightly reduced in comparison to that of the w.t. tetA promoter; both in vivo and in vitro, the transcription start site is outside and downstream the (TA)21 stretch, 5-7 bp upstream that found in the w.t. To adjust the behaviour of B30 and the claimed consensus distance between the E. coli promoter consensus sequences, some structural modification in the (TA)21 stretch -either spontaneous or induced by RNA polymerase- can be hypothesized. Unless the (TA)21 stretch itself plays the role of a relatively good promoter, the results suggest that promoter-specific elements may be distributed along the DNA sequences over distances longer, but seldom less, than the 17 +/- 2 bp consensus distance.

Base Sequence↗
PubMed1983

A microquantitative method for investigating the interaction between labeled lectins and the surface membranes of human lymphocytes using a semi-automatic harvesting machine.

We describe a simple, rapid and economical method for the study of the interaction of labeled lectins and the surface membranes of human lymphocytes using a semi-automatic harvesting machine (Titertek Multiple Cell Harvester). The procedure requires both small numbers of cells and small amounts of lectin, moreover it reduces the number of experimental steps required.

Cell Membrane↗
PubMed1980

Interactions of insolubilized lectins with membrane glycoproteins in presence of detergents.

The effects of several detergents commonly used to solubilize membrane glycoproteins have been investigated on the binding of hepatoma cell surface [3H]-galactoglycoproteins to, and their elution from, concanavalin A or Ricinus communis lectins conjugated to Sepharose 4B. The optimum conditions (pH, ionic strength) in the presence of ionic [sodium deoxycholate (DOC) and sodium dodecyl sulphate (SDS)] and non-ionic detergents (Triton X-100) at a constant concentration were determined in order to ascertain which would yield the better efficiency. The effects of different detergent concentrations on binding and elution were then studied. The range of concentrations for each detergent to be used without modifying efficiency was determined. Triton X-100 and DOC (0.1-1%) did not change the efficiency on Ricinus lectin-Sepharose, whereas SDS, at a concentration greater than 0.05%, caused a dramatic decrease in efficiency. On concanavalin A-Sepharose, by contrast, the non-ionic detergent had no effect on the efficiency at all the concentrations tested (0.1-1%), while concentrations of more than 0.5% DOC and 0.1% SDS significantly decreased both binding and elution.

Animals↗