Biomedical subjects

V Moncollin

Publications and source records attributed to V Moncollin.

31 records · Page 2Linked to original sources

PubMed1992

Class II (B) general transcription factor (TFIIB) that binds to the template-committed preinitiation complex is different from general transcription factor BTF3.

A class II (B) general transcription factor of 34 kDa has been purified from HeLa cells to apparent homogeneity. This factor appears to be transcription factor IIB (TFIIB), since it binds in vitro to template-committed preinitiation complexes formed between a template containing the TATA box/cap-site elements of the adenovirus type 2 major late promoter (Ad2MLP) and recombinant human or yeast TFIID (previously called BTF1) expressed in Escherichia coli. DNase I footprint studies show an extended pattern of protection of Ad2MLP TATA box/cap-site sequences when TFIIB is bound to template-committed complexes, even though TFIIB does not bind on its own to the template in the absence of TFIID. We also show that TFIIB is different from BTF3 by a number of criteria.

Amino Acid Sequence↗

PubMed1992

The acidic transcriptional activator GAL-VP16 acts on preformed template-committed complexes.

The action of the chimeric acidic transcriptional activator GAL-VP16 has been investigated by performing a series of kinetic experiments using the detergent Sarkosyl as well as monoclonal antibodies which specifically inhibit GAL-VP16 DNA binding and transcriptional activation. GAL-VP16 binds to recognition site rapidly, remains bound after transcriptional initiation and is required to maintain stimulated levels of reinitiation. GAL-VP16 action, which appears to result in an increase in the number of preinitiation complexes formed, occurs after the formation of template-committed complexes composed of promoter-bound TFIIA (STF) and a partially purified TFIID fraction conferring GAL-VP16 responsiveness on a reconstituted basal transcription system. This TFIID fraction cannot be replaced by TFIIB or cloned TFIID. Our results suggest that GAL-VP16 activates step(s) in preinitiation complex assembly occurring after TFIID has bound.

Antibodies, Monoclonal↗

PubMed1992

Expression in Escherichia coli: purification and properties of the recombinant human general transcription factor rTFIIB.

The human class II transcription factor TFIIB (rTFIIB) was overexpressed in Escherichia coli using a T7 RNA polymerase expression system and further purified to apparent homogeneity. The purified rTFIIB is identical to the endogenous factor according to the following criteria: molecular weight, microsequencing and mass spectra studies, ability to recognize the stable preinitiation complex formed between TFIID and the adenovirus 2 major late TATA box as demonstrated by gel shift as well as by DNase I footprinting assays, and transcription activity.

Blotting, Western↗

PubMed1991

Purification and interaction properties of the human RNA polymerase B(II) general transcription factor BTF2.

A general transcription factor (BTF2) has been purified from HeLa whole cell extract and shown to be absolutely required for the formation of a functional initiation complex. We also demonstrate that this factor binds in solution to RNA polymerase B(II) and to the other general transcription factors BTF1(TFIID), BTF3, and BTF4. These results strongly suggest the existence of multiple interactions between the various components of the multiprotein initiation complex.

HeLa Cells↗

PubMed1990

A yeast homolog of the human UEF stimulates transcription from the adenovirus 2 major late promoter in yeast and in mammalian cell-free systems.

We report the identification and purification of a yeast factor functionally homologous to the human upstream element factor (UEFh). Although the yeast protein (UEFy) has a higher molecular weight than the HeLa UEF (60 kD versus 45 kD) both have identical DNA-binding properties: the purified UEFy recognizes the Adenovirus 2 (Ad2) major late promoter upstream element (MLP-UE; from nucleotide -49 to -67) as well as the IVa2 upstream element (IVa2-UE; from nucleotide -98 to -122) with a higher affinity for the MLP-UE than for the IVa2-UE. Based on its DNA binding specificity, size and thermostability, the UEFy protein appears also similar or equivalent to the centromere binding protein CP1. In a competition assay with oligonucleotides containing the MLP-UE binding site, a drastic reduction of Ad2 MLP transcription was observed both in a HeLa and in a yeast cell free system, which was restored by addition of either the purified UEFh or UEFy proteins. We conclude that both UEFh and UEFy activate transcription from the Ad2 MLP upon binding to the upstream element, whatever is the in vitro cell-free system (yeast or HeLa). This indicate that some regulatory function represented by the upstream element and its cognate factor, is well conserved between human and yeast.

Adenoviruses, Human↗

PubMed1990

Purification of the upstream element factor of the adenovirus-2 major late promoter from HeLa and yeast by sequence-specific DNA affinity chromatography.

The purification to homogeneity of the Adenovirus-2 major late promoter (MLP) upstream element factor (UEF), a sequence specific transcription factor, which binds to upstream elements of various class B (II) genes, is reported. The protein was purified from HeLa cells and also from the yeast Saccharomyces cerevisiae, by using sequence-specific DNA affinity chromatography. The human (UEFh, 45,000 dalton) and the yeast (UEFy, 60,000 dalton) proteins protect the same sequences over the MLP-IVa2 intergenic region: the MLP-UE (from nucleotide -49 to -67) and the IVa2-UE (from nucleotide -98 to -122 relative to the MLP initiation site). Both proteins have a higher affinity for the MLP-UE than for the IVa2-UE.

Adenoviruses, Human↗

PubMed1990

The mammalian upstream element factor recognizes two sites in the adenovirus type 2 IVa2-major late promoter intergenic region and stimulates both promoters.

The adenovirus type 2 major late upstream element factor (UEF) recognizes two similar elements that lie between the major late promoter (MLP) and IVa2 promoter cap sites (the previously characterized MLP-UE from nucleotides -49 to -67 and the IVa2-UE from nucleotides -98 to -122). DNase I footprinting and gel retention assays showed that the UEF has a lower affinity for the IVa2-UE than for the MLP-UE. In vitro transcription experiments demonstrated first that the IVa2 promoter, which lacks a consensus TATA box, may work, as does the MLP, in the absence of its proximal upstream element and second that the IVa2-UE stimulated IVa2 transcription two- to threefold, as MLP-UE did for the MLP. In addition, we demonstrated that the more distal upstream element has a weak stimulatory effect on transcription of both promoters.

Adenoviruses, Human↗

PubMed1987

A general transcription factor forms a stable complex with RNA polymerase B (II).

A general transcription factor (BTF3) has been purified from HeLa whole cell extracts and shown to be required for accurate initiation of transcription from the adenovirus-2 major late promoter (Ad2MLP) and other RNA polymerase class B promoters. We show that purified BTF3 (27 kd) binds to RNA polymerase B (II), forming a complex that is transcriptionally active. We found no evidence that purified BTF3 interacts with DNA or is required for the formation of the stable preinitiation complex.

Adenoviridae↗

PubMed1986

Purification of a factor specific for the upstream element of the adenovirus-2 major late promoter.

Stimulation of in vitro transcription by the upstream element (UE) of the adenovirus-2 major late promoter (Ad2MLP) involves a specific trans-acting factor present in a HeLa whole-cell extract. By following its transcriptional stimulatory activity and its DNase I footprint on the Ad2MLP-UE, we have purified this factor to greater than 10% purity and separated it from RNA polymerase B and the general transcription factors required for transcription from the Ad2MLP.

Adenoviruses, Human↗

PubMed1985

Specific interaction between a transcription factor and the upstream element of the adenovirus-2 major late promoter.

Stimulation of in vitro transcription mediated by the upstream element of the adenovirus-2 major late promoter (Ad2MLP) involves its recognition by a specific trans-acting factor present in a HeLa whole-cell extract. DNase I footprinting and dimethylsulfate methylation protection experiments were used to determine, at the nucleotide level, upstream sequences which interact with this transcription factor. The ability of upstream element mutants to bind the transcription factor correlates directly with the efficiency of transcription from the corresponding Ad2ML promoters in vivo and in vitro. Competition footprinting experiments show that the transcription factor, which binds to the upstream element of the Ad2MLP, can also interact, but with a lower affinity, with the upstream elements of the Ad2E2a and rabbit beta-globin promoters, both of which display some sequence homology to the 'interacting' region of the Ad2MLP upstream element. The transcription factor does not, however, interact with the upstream elements of either the Ad2E2L, Ad5E3, SV40 early, herpes virus thymidine kinase or chicken conalbumin promoters.

Adenoviruses, Human↗

PubMed1984

Stimulation of in vitro transcription by the upstream element of the adenovirus-2 major late promoter involves a specific factor.

We have previously reported that sequences located upstream from the TATA box of the Adenovirus-2 major late promoter (Ad2MLP), between -34 and -97, are necessary for efficient transcription in vivo and in vitro (1). We have utilized an in vitro competition assay to demonstrate that the upstream element requirement involves the binding of a specific factor(s), which results in stimulation of in vitro transcription from the Ad2MLP. DNA fragments prepared from Ad2MLP upstream sequence mutants which are transcribed much less efficiently than the wild-type promoter both in vivo and in vitro were shown to be unable to bind this factor. We have also constructed chimeric promoter recombinants containing the 21bp repeat upstream element of the SV40 early promoter inserted upstream from the Ad2MLP TATA box. The SV40 upstream element stimulates in vitro transcription from the heterologous Ad2MLP TATA box element; competition experiments show that the Ad2MLP upstream element-specific factor is different from the SV40-specific factor Sp1.

Adenoviruses, Human↗

PubMed1984

Is actin a transcription initiation factor for RNA polymerase B?

We have previously reported that two fractions derived from HeLa cell S100 extracts, the heparin flow-through and the heparin 0.6 M KCl eluate are required in vitro for efficient and accurate transcription by RNA polymerase class B (II). We have further purified a factor present in the heparin flow-through fraction, which markedly stimulates specific transcription catalyzed by the heparin 0.6 M KCl eluate. We report here that some of the properties of the stimulatory factor present in our most purified fractions are strikingly similar to those of actin. We demonstrate also that this factor acts at the pre-initiation level of the transcription reaction.

Actins↗

PubMedDate not supplied

Expression in Escherichia coli: purification and properties of the yeast general transcription factor TFIID.

A T7 RNA polymerase expression system has been used for the efficient expression of the yeast RNA polymerase general transcription factor TFIID (TFIIDY), the TATA-box factor (previously called BTF1) in Escherichia coli. Expression of the gene was performed at 25 degrees C instead of 37 degrees C to increase the total amount of soluble TFIIDY. Soluble TFIIDY was purified in three chromatographic steps and was eluted from the final column, a heparin-5PW HPLC column, in two peaks at 0.38 M (peak I) and 0.42 M (peak II) KCl in which this protein was 52% and greater than 95% pure, respectively. The protein in both peaks was active in an in vitro transcription assay. However, while TFIIDY from peak II was essentially indistinguishable from the material isolated from yeast, the protein of peak I differed in a number of biochemical characteristics, having a lower specific activity in an in vitro transcription assay and displaying an altered pattern of bands in a DNA band shift assay. Despite these differences, the proteins in both peaks have identical molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, have indistinguishable N-terminal amino acid sequences, and apparently exist as monomers under the conditions used for the heparin-5PW chromatography.

Amino Acid Sequence↗