Biomedical subjects

G Duval-Valentin

Publications and source records attributed to G Duval-Valentin.

9 recordsLinked to original sources

PubMed1995

Triple-helix specific ligands stabilize H-DNA conformation.

Under superhelical stress, oligopurine-oligopyrimidine mirror-repeat sequences are able to adopt H-DNA conformations where a triple-helical and a single-stranded structure co-exist. We have previously shown that a benzo[e]pyridoindole derivative (BePI), an antitumor drug interacting more tightly with triplex than with duplex DNA, strongly stabilizes intermolecular triple helices formed upon binding of homopyrimidine oligonucleotides to the major groove of double-stranded DNA at oligopurine-oligopyrimidine sequences. Here we show that an intramolecular triple helix is also strongly stabilized by this ligand. In vitro elongation performed by different DNA polymerases (bacteriophage T7, Escherichia coli or Taq polymerase) could be irreversibly inhibited by the H-DNA structure in the presence of BePI. A mirror-repeat polypurine-polypyrimidine sequence inserted between the E. coli beta-lactamase gene (conferring ampicillin resistance) and its bla promoter strongly inhibited transcription of the beta-lactamase gene in vivo. In the absence of supercoiling, transition to the H-conformation did not occur, but BePI stabilized the H-DNA structure induced by supercoiling as shown by chemical probes (chloroacetaldehyde). The results presented here open a new field of investigation for antitumor agents targeted to a novel class of genetic structures able to regulate gene expression.

Base Sequence↗

PubMed1992

Triple helix-specific ligands.

A triple helix is formed upon binding of an oligodeoxynucleotide to the major groove of duplex DNA. A benzo[e]pyridoindole derivative (BePI) strongly stabilized this structure and showed preferential binding to a triplex rather than to a duplex. Energy transfer experiments suggest that BePI intercalates within the triple helix. Sequence-specific inhibition of transcription initiation of a specific gene by Escherichia coli RNA polymerase by a triplex-forming oligodeoxynucleotide is strongly enhanced when the triplex is stabilized by BePI. Upon irradiation with ultraviolet light, BePI induces covalent modifications of the target within the triple helix structure.

Base Sequence↗

PubMed1992

Specific inhibition of transcription by triple helix-forming oligonucleotides.

Homopyrimidine oligonucleotides bind to the major groove of a complementary homopyrimidine.homopurine stretch by triple helix formation. The bla gene from transposon Tn3 contains a homopyrimidine.homopurine sequence of 13 base pairs located just downstream of the RNA polymerase binding site. A 13-mer homopyrimidine oligonucleotide targeted to this sequence was tested for its effect on transcription of the bla gene in vitro. We show that the consequence of triple helix formation in front of the Escherichia coli RNA polymerase-promoter complex is to block the holoenzyme at its start site during a period that is dependent on temperature. The temperature dependence of transcription inhibition shows a direct correlation between this effect and the stabilization of the triple helix. Substitution of 5-methylcytosine to cytosine in the 13-mer oligonucleotide enhances triplex stability and transcription inhibition. Transcription inhibition by this synthetic repressor was also confirmed by footprinting studies demonstrating its specificity of action. The 13-mer oligonucleotide containing a psoralen derivative covalently linked to its 5' end shows an irreversible and specific inhibition of transcription initiation after exposure to light of wavelength greater than 310 nm.

Base Sequence↗

PubMed1991

Extension of the range of recognition sequences for triple helix formation by oligonucleotides containing guanines and thymines.

Oligodeoxynucleotides containing G and T can bind to homopurine.homopyrimidine sequences on double-stranded DNA by forming C.G x G and T.A x T base triplets. The orientation of the third strand in such triple helices depends on the number of GpT and TpG steps. Therefore a single oligonucleotide can be designed to bind to two consecutive homopurine.homopyrimidine sequences where the two homopurine stretches alternate on the two strands of DNA. The oligonucleotide switches from one homopurine strand to the other at the junction between the two sequences. This result shows that it is possible to extend the range of DNA sequences that can be recognized by a single oligonucleotide.

Base Sequence↗

PubMed1990

How Escherichia coli RNA polymerase can negatively regulate transcription from a constitutive promoter.

We previously described the structures and functions of specific complexes between the bla promoter from Tn3 (present in pBR322) and RNA polymerase (RNAP), showing that, at excess RNAP, complexes can form in which one or two RNAPs bind to the same promoter (1:1 and 2:1 complexes) (Duval-Valentin and Ehrlich, 1988). We report here that the 2:1 complex cannot be detected below 25 degrees C; above that temperature, a 1:1 complex forms at a rate one order of magnitude faster than that of the 2:1 complex, and above 30 degrees C, the amounts of both species become equal for RNAP/promoter ratio r30 less than or equal to r less than or equal to 70. The 2:1 complex decays back to a 1:1 complex losing the last RNAP at a rate about three times that of the 1:1 complex decay. Functional assays of the complexes formed at excess RNAP show that both 1:1 and 2:1 complexes are immediately and permanently inhibited, even when the promoters are pre-incubated with ribonucleotide selections potentially enabling entrance into abortive cycling or formation of a stressed complex. We conclude that the inhibition step probably takes place in the complex formation pathway between RPi and RPo, at a novel stable intermediate isomer, RPj, formed above 25 degrees C. A possible mechanism of formation of the 2:1 complex is outlined. In vivo studies, in which r was modified by varying the bacterial growth rate, show a reduction of bla expression as r values are upshifted, specific to the bla promoter from Tn3.

DNA-Directed RNA Polymerases↗

PubMed1988

A second RNA-polymerase can bind specifically to the bla promoter of Tn3, repressing transcription initiation.

We showed earlier that the region of the bla promoter of Tn3 protected by the RNA-polymerase (RNAP), has the normal size (about 60bp) at RNAP/promoter molar ratio r less than or equal to 2, but rises to about twice this extent as r increases. We confirm here that the species corresponding to normal and extended footprint distinguish by their electrophoretic mobilities. Furthermore, inspection of the complexes by electron microscopy confirms that at r greater than 2, the bla promoter can bind specifically a second RNAP particle, as compared to the 1:1 complex observed at r less than or equal to 2. At r greater than 2, the ability of the bla promoter to initiate transcription in vitro is repressed when compared to the complex 1:1 obtained at r less than or equal to 2. The unexpected decrease in initiation efficiency as the concentration of RNAP particles is increased, together with the striking sequence homology of the bla promoter with promoters of stable RNA, suggest that in vivo, this promoter could be regulated by growth rate.

DNA Transposable Elements↗

PubMed1988

Far upstream sequences of the bla promoter from TN3 are involved in complexation with E. coli RNA-polymerase.

The structure of the final initiation complex between E. coli RNA polymerase (RNAP) and the bla promoter from the transposon TN3 has been probed by footprinting experiments and base accessibility to dimethyl sulfate at 37 degrees C. At RNAP/promoter molar ratios "standard" for these experiments (greater than or equal to 10), the contacts on bla extend from -100 to +20, i.e. a length exceeding twice the dimension of the RNAP major axis [33]. Since footprinting at about equimolar amounts of RNAP and bla extends to the usual (-55 to +20) promoter domain, it is very likely that at least two RNAP's participate in the complex observed at tenfold higher RNAP/bla ratios. Under the latter conditions, the extended footprint (-100 to +20) is observed above 30 degrees C, whereas at 15 degrees C, only the -55 to +20 promoter area is contacted. Furthermore, gel retardation experiments show the presence of two complexes of different migration rates. We have reported earlier [21] that at the "standard" RNAP/bla ratio, transcription initiation from the bla promoter is inhibited. The correlation of this inhibition with the postulated two RNAP/bla complex suggests a regulation of bla gene expression by RNAP availability controlled for instance by growth rate. These results can be correlated with those reported in [14, 15] for the tyrT promoter. Interestingly, both promoter share significant sequence homologies.

DNA Transposable Elements↗

PubMed1987

Dynamic and structural characterisation of multiple steps during complex formation between E. coli RNA polymerase and the tetR promoter from pSC101.

Kinetic, functional and structural studies of the recognition of the tetR promoter from pSC101 by E. coli RNA polymerase allowed the characterization of several steps in the specific complex formation and transcription initiation process. First, enzyme and DNA enter in a short life-time complex. An isomerization will convert this unstable complex into a closed stable one where RNA polymerase is tightly attached without establishing stable chemical contacts with the bases. In the next step, stable close contacts appear between both macromolecules involving mainly the downstream part of the promoter. A further isomerization will lead to an open complex where DNA is locally melted and the system is able to initiate transcription. This latter process is accompanied by changes in the upstream part of the promoter. Finally, in vitro transcription assays showed that the position of the major transcription start sites depends on temperature. From the reported results, it appears that the recognition event is a sequential process where different structural elements of the promoter, that can be located apart in the sequence, are involved in a concerted manner in each stage.

Cytosine↗

PubMed1986

Interaction between E. coli RNA polymerase and the tetR promoter from pSC101: homologies and differences with other E. coli promoter systems from close contact point studies.

The interaction between E. coli RNA polymerase and the tetR promoter from pSC101, was studied by protection and premodification experiments, using dimethyl sulfate, methylation of single stranded cytosines, and DNAase I footprinting. Whereas qualitative and quantitative results from the chemical approach conform to patterns already displayed by other promoter systems, hypersensitive sites to DNAase I attack differ from those of other promoters. Distribution and nature of the contacts suggest that regions of the promoter sequence participates differently in complex formation. The involvement of major and minor grooves of the double helix in the complex with the enzyme, differs along the promoter. After a comparison of the results from seven different promoters, a pattern of conserved contacts seem to appear. Comparison of temperature dependence of local unwinding around the transcription start site (detected by the appearance of single stranded cytosines), and DNAase I footprinting, reveals that the process leading to stable complex formation can be achieved without disruption of base-pairing.

Base Sequence↗