scholarly journals Yeast transcription factor IID participates in cell-free transcription of a mammalian ribosomal protein TATA-less promoter

1992 ◽  
Vol 285 (3) ◽  
pp. 721-723 ◽  
Author(s):  
T Yoganathan ◽  
M Horikoshi ◽  
S Hasegawa ◽  
R G Roeder ◽  
B H Sells
Keyword(s):  
Transcription Factor ◽  
Ribosomal Protein ◽  
Transcription Initiation ◽  
Nuclear Extract ◽  
Nuclear Extracts ◽  
Alternative Transcription ◽  
Before And After ◽  
Transcription Factor Iid ◽  
Tata Sequence

We analysed transcription of the gene for the ribosomal protein (rp) L32 of the mouse, which is transcribed in mouse L1210 nuclear extracts in vitro. The rpL32 gene lacks a canonical TATA box. Hence it has been suggested that this gene has an alternative transcription pathway not requiring transcription factor IID (TFIID). Selective inactivation of TFIID in nuclear extract completely abolished the transcription of rpL32 in vitro. Selective inactivation was restored by the addition of cloned and purified yeast TFIID (yTFIID), indicating that this TATA-less rpL32 promoter utilizes TFIID for its transcription initiation. Furthermore, addition of an oligonucleotide-containing TATA sequence interfered with the rpL32 transcription and this was overcome by the addition of yTFIID. To further examine the stage of involvement of TFIID in rpL32 transcription, TATA oligonucleotide was added to nuclear extract before and after the formation of the transcription complex. The results reveal that TFIID associates with the pre-initiation complex and that this complex is largely resistant to added TATA oligonucleotide. Our results show, for the first time, that the TATA-less rpL32 gene utilizes TFIID for transcription initiation.

Identification of promoter elements necessary for transcriptional regulation of a human histone H4 gene in vitro

1985 ◽  
Vol 5 (2) ◽  
pp. 380-389
Author(s):  
S M Hanly ◽  
G C Bleecker ◽  
N Heintz
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Initiation Site ◽  
S Phase ◽  
Histone H4 ◽  
Promoter Elements ◽  
Nuclear Extracts ◽  
Distal Promoter ◽  
Histone H4 Gene

We have examined the nucleotide sequences necessary for transcription of a human histone H4 gene in vitro. Maximal transcription of the H4 promoter requires, in addition to the TATA box and cap site, promoter elements between 70 and 110 nucleotides upstream from the transcription initiation site. These distal promoter elements are recognized preferentially in extracts from synchronized S-phase HeLa cells. The inability of non-S-phase nuclear extracts to recognize the H4 upstream sequences reflects a specific lack of a transcription factor which interacts with those sequences. These results indicate that the cell cycle regulation of human histone gene expression involves both a specific transcription factor and distal transcription signals in the H4 promoter.

scholarly journals Identification of promoter elements necessary for transcriptional regulation of a human histone H4 gene in vitro.

1985 ◽  
Vol 5 (2) ◽  
pp. 380-389 ◽  
Author(s):  
S M Hanly ◽  
G C Bleecker ◽  
N Heintz
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Initiation Site ◽  
S Phase ◽  
Histone H4 ◽  
Promoter Elements ◽  
Nuclear Extracts ◽  
Distal Promoter ◽  
Histone H4 Gene

We have examined the nucleotide sequences necessary for transcription of a human histone H4 gene in vitro. Maximal transcription of the H4 promoter requires, in addition to the TATA box and cap site, promoter elements between 70 and 110 nucleotides upstream from the transcription initiation site. These distal promoter elements are recognized preferentially in extracts from synchronized S-phase HeLa cells. The inability of non-S-phase nuclear extracts to recognize the H4 upstream sequences reflects a specific lack of a transcription factor which interacts with those sequences. These results indicate that the cell cycle regulation of human histone gene expression involves both a specific transcription factor and distal transcription signals in the H4 promoter.

High resolution mapping of nucleic acid containing complexes in vitro and in situ

1996 ◽  
Vol 54 ◽  
pp. 48-49
Author(s):  
D. P. Bazett-Jones ◽  
M. J. Hendzel
Keyword(s):  
Nucleic Acid ◽  
High Resolution ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Heavy Atom ◽  
Regulation Of Transcription ◽  
High Exposure ◽  
Resolution Mapping ◽  
Tata Sequence

Structural analysis of combinations of nucleosomes and transcription factors on promoter and enhancer elements is necessary in order to understand the molecular mechanisms responsible for the regulation of transcription initiation. Such complexes are often not amenable to study by high resolution crystallographic techniques. We have been applying electron spectroscopic imaging (ESI) to specific problems in molecular biology related to transcription regulation. There are several advantages that this technique offers in studies of nucleoprotein complexes. First, an intermediate level of spatial resolution can be achieved because heavy atom contrast agents are not necessary. Second, mass and stoichiometric relationships of protein and nucleic acid can be estimated by phosphorus detection, an element in much higher proportions in nucleic acid than protein. Third, wrapping or bending of the DNA by the protein constituents can be observed by phosphorus mapping of the complexes. Even when ESI is used with high exposure of electrons to the specimen, important macromolecular information may be provided. For example, an image of the TATA binding protein (TBP) bound to DNA is shown in the Figure (top panel). It can be seen that the protein distorts the DNA away from itself and much of its mass sits off the DNA helix axis. Moreover, phosphorus and mass estimates demonstrate whether one or two TBP molecules interact with this particular promoter TATA sequence.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

Multiple hepatic trans-acting factors are required for in vitro transcription of the human alpha-1-antitrypsin gene

1988 ◽  
Vol 8 (10) ◽  
pp. 4362-4369
Author(s):  
Y Li ◽  
R F Shen ◽  
S Y Tsai ◽  
S L Woo
Keyword(s):  
Rat Liver ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Flanking Sequence ◽  
Tissue Specific ◽  
Hepatic Expression ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Alpha 1 Antitrypsin

The human alpha-1-antitrypsin (AAT) gene is expressed in the liver, and its deficiency causes pulmonary emphysema. We have demonstrated that its 5'-flanking region contains cis-acting elements capable of directing proper transcription in the presence of rat liver nuclear extract. The in vitro transcription system is tissue-specific in that the AAT promoter is functional in nuclear extracts prepared from the liver but not from HeLa cells. Experiments in which rat liver and HeLa nuclear extracts were mixed suggested the presence of a specific activator(s) in hepatocytes rather than a repressor(s) in nonproducing cells. Two protected regions were detected in the promoter by DNase I footprinting analysis with rat liver nuclear extracts. Region one spanned -78 to -52 and region two spanned -125 to -100 in the 5'-flanking sequence of the gene. By gel retardation assays with synthetic oligonucleotides, at least two distinct liver nuclear factors were identified, HNF-1 and HNF-2 (hepatocyte nuclear factors), which bound specifically to the first and second region, respectively. We present evidence that HNF-1 and HNF-2 are positively acting, tissue-specific transcription factors that regulate hepatic expression of the human AAT gene.

Two transcriptional activators, CCAAT-box-binding transcription factor and heat shock transcription factor, interact with a human hsp70 gene promoter

1987 ◽  
Vol 7 (3) ◽  
pp. 1129-1138
Author(s):  
W D Morgan ◽  
G T Williams ◽  
R I Morimoto ◽  
J Greene ◽  
R E Kingston ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Gene Promoter ◽  
Heat Shock Transcription Factor ◽  
Transcriptional Activators ◽  
Hsp70 Gene ◽  
Nuclear Extracts ◽  
Ccaat Box ◽  
Human Hsp70

We characterized the activity of a human hsp70 gene promoter by in vitro transcription. Analysis of 5' deletion and substitution mutants in HeLa nuclear extracts showed that the basal activity of the promoter depends primarily on a CCAAT-box sequence located at -65. A protein factor, CCAAT-box-binding transcription factor (CTF), was isolated from HeLa nuclear extracts and shown to be responsible for stimulation of transcription in a reconstituted in vitro system. DNase I footprinting revealed that CTF interacts with two CCAAT-box elements located at -65 and -147 of the human hsp70 promoter. An additional binding activity, heat shock transcription factor (HSTF), which interacted with the heat shock element, was also identified in HeLa extract fractions. This demonstrates that the promoter of this human hsp70 gene interacts with at least two positive transcriptional activators, CTF, which is required for CCAAT-box-dependent transcription as in other promoters such as those of globin and herpes simplex virus thymidine kinase genes, and HSTF, which is involved in heat inducibility.

UV cross-linking identifies four polypeptides that require the TATA box to bind to the Drosophila hsp70 promoter

1990 ◽  
Vol 10 (8) ◽  
pp. 4233-4238
Author(s):  
D S Gilmour ◽  
T J Dietz ◽  
S C Elgin
Keyword(s):  
Specific Protein ◽  
Cross Linking ◽  
Dependent Manner ◽  
Hsp70 Promoter ◽  
Intimate Contact ◽  
Nuclear Extracts ◽  
Multicomponent Complex ◽  
Tata Sequence ◽  
Drosophila Embryos

A protein fraction that requires the TATA sequence to bind to the hsp70 promoter has been partially purified from nuclear extracts of Drosophila embryos. This TATA factor produces a large DNase I footprint that extends from -44 to +35 on the promoter. A mutation that changes TATA to TATG interferes both with the binding of this complex and with the transcription of the hsp70 promoter in vitro, indicating that this interaction is important for transcriptional activity. Using a highly specific protein-DNA cross-linking assay, we have identified four polypeptides that require the TATA sequence to bind to the hsp70 promoter. Polypeptides of 26 and 42 kilodaltons are in intimate contact with the TATA sequence. Polypeptides of 150 and 60 kilodaltons interact within the region from +24 to +47 in a TATA-dependent manner. Both the extended footprint and the polypeptides identified by UV cross-linking indicate that the Drosophila TATA factor is a multicomponent complex.

Cytoplasmic and nuclear progesterone receptors in mouse mammary tumours during pregnancy determined by an improved hydroxylapatite assay

1982 ◽  
Vol 94 (1) ◽  
pp. 111-123 ◽  
Author(s):  
Y. Koseki ◽  
M. E. Costlow ◽  
D. Cole ◽  
A. Matsuzawa
Keyword(s):  
Progesterone Receptors ◽  
Nuclear Extract ◽  
Scatchard Analysis ◽  
Normal Mammary Gland ◽  
Single Class ◽  
Heat Stable ◽  
Nuclear Extracts ◽  
Complete Exchange

The binding of [3H] 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (R5020) to progesterone receptors in cytosol and nuclear extracts (0·6 m-KCl) of the pregnancy-dependent, TPDMT-4 mouse mammary tumour was measured at various stages of pregnancy. Compared with conventional dextran-coated charcoal (DCC) assays, a hydroxylapatite assay with DCC pretreatment and precharging of the cytosol with unlabelled R5020 (4 × 10−8 mol/l, for 3–4 h at 4 °C) showed the highest level of binding. The DCC treatment markedly increased the level of R5020 binding in both cytosol and nuclear extracts by allowing the receptor to bind to hydroxylapatite. The DCC pretreatment apparently removed a heat-stable and non-dialysable factor which prevented the receptor from binding to the hydroxylapatite. Using this assay R5020 binding reached a steady state in 24 h at 4 °C, with complete exchange of radioactive for non-radioactive ligand by 20 h. Nuclear extracts did not require precharging and complete exchange was more rapid. Scatchard analysis (without precharging) disclosed a single class of binding sites with a dissociation constant for cytosol of 3·2 ± 0·8 (s.e.m.) × 10−9 mol/l (n = 3) and 4·7 ± 0·6 × 10−9 mol/l (n = 5) for the nuclear extract. Binding was hormone-specific and progesterone translocated binding from the cytoplasm to the nucleus both in vivo and in vitro. Translocation, however, led to a substantial loss of total (nuclear + cytoplasmic receptors. During pregnancy, cytoplasmic progesterone receptor levels were unchanged and low compared to nuclear progesterone receptors which increased by sevenfold from days 1 to 11 and then decreased at day 16. Compared with recent data on cytoplasmic progesterone receptors in normal mammary gland, our results suggested that this tumour may have a reduced sensitivity to the down-regulatory activity of progesterone. This lesion may, in part, account for the failure of the tumour to differentiate during pregnancy.

scholarly journals The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor.

1994 ◽  
Vol 14 (1) ◽  
pp. 373-381 ◽  
Author(s):  
D E Zhang ◽  
C J Hetherington ◽  
H M Chen ◽  
D G Tenen
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Transcription Initiation ◽  
Colony Stimulating Factor ◽  
Specific Expression ◽  
Tissue Specific ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The macrophage colony-stimulating factor (M-CSF) receptor is expressed in a tissue-specific fashion from two distinct promoters in monocytes/macrophages and the placenta. In order to further understand the transcription factors which play a role in the commitment of multipotential progenitors to the monocyte/macrophage lineage, we have initiated an investigation of the factors which activate the M-CSF receptor very early during the monocyte differentiation process. Here we demonstrate that the human monocytic M-CSF receptor promoter directs reporter gene activity in a tissue-specific fashion. Since one of the few transcription factors which have been implicated in the regulation of monocyte genes is the macrophage- and B-cell-specific PU.1 transcription factor, we investigated whether PU.1 binds and activates the M-CSF receptor promoter. Here we demonstrate that both in vitro-translated PU.1 and PU.1 from nuclear extracts bind to a specific site in the M-CSF receptor promoter just upstream from the major transcription initiation site. Mutations in this site which eliminate PU.1 binding decrease M-CSF receptor promoter activity significantly in macrophage cell lines only. Furthermore, PU.1 transactivates the M-CSF receptor promoter in nonmacrophage cells. These results suggest that PU.1 plays a major role in macrophage gene regulation and development by directing the expression of a receptor for a key macrophage growth factor.

Sign in / Sign up

Export Citation Format

Share Document