scholarly journals Novel Activation Step Required for Transcriptional Competence of Progesterone Receptor on Chromatin Templates

2003 ◽  
Vol 17 (12) ◽  
pp. 2543-2553 ◽  
Author(s):  
Varykina G. Thackray ◽  
David O. Toft ◽  
Steven K. Nordeen
Keyword(s):  
Progesterone Receptor ◽  
Transcriptional Activity ◽  
Specific Binding ◽  
Specific Inhibitor ◽  
Transcription System ◽  
Naked Dna ◽  
Activation Of Transcription ◽  
Chromatin Template ◽  
Reticulocyte Lysate ◽  
A Cell

Abstract To elucidate the earliest molecular steps in the activation of transcription by the progesterone receptor (PR), we investigated its activity in a cell-free transcription system utilizing chromatin templates. PR prepared as a ligand-free, recombinant protein failed to induce transcription on chromatin templates. However, transcriptional competence could be restored by coincubation with rabbit reticulocyte lysate (RRL). The interaction of PR with chaperones results in a receptor conformation competent to bind ligand and RRL contains abundant chaperone-mediated protein folding activity. Blocking this activity with the specific inhibitor geldanamycin inhibited receptor-dependent transcriptional activity. However, recombinant chaperones could not replace RRL in the restoration of transcriptional activity on chromatin templates, suggesting the presence of an additional activity in the lysate. Under chromatin assembly conditions, PR could bind naked DNA and RRL did not increase that binding. In contrast, PR bound to a chromatin template only poorly. Interestingly, RRL stimulated sequence-specific binding by PR to target sites in chromatin and the concomitant recruitment of the steroid receptor coactivator 1 to the promoter. Thus, our results indicate that a novel protein-mediated activity in RRL is involved in an additional, heretofore unrecognized, activation step required for PR to become transcriptionally competent on chromatin templates.

Effect of in vivo oestradiol treatment on cell-free transcription in trout liver nuclear extracts

1994 ◽  
Vol 13 (2) ◽  
pp. 137-147 ◽  
Author(s):  
F Delaunay ◽  
F Pakdel ◽  
Y Valotaire
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activity ◽  
Hormonal Treatment ◽  
Fold Increase ◽  
Flanking Sequence ◽  
Transcription System ◽  
Nuclear Extracts ◽  
B1 Gene ◽  
A Cell

ABSTRACT In order to perform later studies on the transcriptional regulation of hormone-dependent genes in fish liver, we firstly examined the potential of trout liver nuclear extracts in a cell-free transcription system. As reporter genes, we used DNA sequences without G (G-free cassettes) under the control of three promoters derived from the 5′ flanking sequence of the Xenopus vitellogenin B1 gene; two of them were responsive to the oestrogen receptor (ER) through oestrogen responsive elements (ERE). Maximal transcriptional activity was obtained within a range of 40–130 μg protein per extract depending on the extract preparation. Transcription was maximal in reactions carried out at 25 °C. Similar transcriptional activities for the three promoters were observed when transcription was performed in extracts from untreated male trout. In contrast, we observed a 4·5- to 6-fold increase in the transcription with ERE-containing promoters in comparison with that with the minimal promoter bearing only a TATA box when extracts from oestradiol-treated male trout were used. This effect was correlated with the increase in the nuclear ER concentration induced by in vivo hormonal treatment. This enhanced transcription was specifically inhibited by the addition of a 25- to 100-fold excess of ERE oligonucleotide competitor. These data demonstrated, therefore, that transcription was ERE-dependent in this system and suggest strongly that it was mediated by the trout ER. Addition of oestradiol or the anti-oestrogens hydroxytamoxifen or ICI 164384 had no effect on the transcriptional activity of the two ERE-containing promoters, indicating that transcription was hormone-independent in trout liver nuclear extracts.

scholarly journals Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70

1993 ◽  
Vol 13 (2) ◽  
pp. 869-876
Author(s):  
D F Smith ◽  
W P Sullivan ◽  
T N Marion ◽  
K Zaitsu ◽  
B Madden ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Simian Virus 40 ◽  
Immunoblot Analysis ◽  
Simian Virus ◽  
Rabbit Reticulocyte Lysate ◽  
Striking Similarity ◽  
Peptide Fragments ◽  
Receptor Complexes ◽  
Reticulocyte Lysate ◽  
A Cell

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.

scholarly journals Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

1993 ◽  
Vol 13 (2) ◽  
pp. 869-876 ◽  
Author(s):  
D F Smith ◽  
W P Sullivan ◽  
T N Marion ◽  
K Zaitsu ◽  
B Madden ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Simian Virus 40 ◽  
Immunoblot Analysis ◽  
Simian Virus ◽  
Rabbit Reticulocyte Lysate ◽  
Striking Similarity ◽  
Peptide Fragments ◽  
Receptor Complexes ◽  
Reticulocyte Lysate ◽  
A Cell

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.

scholarly journals Power-law behavior of transcription factor dynamics at the single-molecule level implies a continuum affinity model

2021 ◽  
Author(s):  
David A Garcia ◽  
Gregory Fettweis ◽  
Diego M Presman ◽  
Ville Paakinaho ◽  
Christopher Jarzynski ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Power Law ◽  
Dwell Time ◽  
Specific Binding ◽  
Power Law Distribution ◽  
Single Molecule Level ◽  
Binding Behavior ◽  
Chromatin Template ◽  
Nuclear Domains

Abstract Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs—one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). Here, we present an analytical pipeline to evaluate which model best explains SMT data. We find that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we propose a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template.

scholarly journals Truncated APC regulates the transcriptional activity of β-catenin in a cell cycle dependent manner

2006 ◽  
Vol 16 (2) ◽  
pp. 199-209 ◽  
Author(s):  
Jean Schneikert ◽  
Annette Grohmann ◽  
Jürgen Behrens
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activity ◽  
Dependent Manner ◽  
A Cell ◽  
Cycle Dependent

Pausing and termination of human RNA polymerase II transcription at a procaryotic terminator

1983 ◽  
Vol 3 (10) ◽  
pp. 1687-1693
Author(s):  
G W Hatfield ◽  
J A Sharp ◽  
M Rosenberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Sequence Specificity ◽  
Late Promoter ◽  
Transcription System ◽  
A Cell ◽  
Major Late Promoter ◽  
Dyad Symmetry ◽  
Rna Polymerase Ii Transcription ◽  
Terminator Sequence

Kinetic analyses of runoff transcription in a cell-free eucaryotic transcription system revealed that the bacteriophage lambda 4S RNA terminator caused human RNA polymerase II to pause on the template and partially terminate transcription of transcripts initiated by the adenovirus 2 major late promoter. Analogous to the procaryotic RNA polymerase, the eucaryotic enzyme terminated just beyond the guanine-plus-cytosine-rich region of dyad symmetry in the terminator sequence. These results suggest that the eucaryotic RNA polymerase II may respond to transcription termination sequences similar to those used by the procaryotic enzyme. However, similar templates containing lambda tint or lambda tR1 terminators did not elicit pausing or termination, suggesting that other features, such as sequence specificity, may also be involved.

A heat-labile factor promotes premature 3' end formation in exon 1 of the murine adenosine deaminase gene in a cell-free transcription system

1991 ◽  
Vol 11 (11) ◽  
pp. 5398-5409
Author(s):  
J W Innis ◽  
R E Kellems
Keyword(s):  
Steady State ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Adenosine Deaminase ◽  
Free System ◽  
Cell Free System ◽  
Transcription System ◽  
Exon 1 ◽  
A Cell ◽  
Processed Products

An elongation block to RNA polymerase II transcription in exon 1 is a major regulatory step in expression of the murine adenosine deaminase (ADA) gene. Previous work in the laboratory identified abundant short transcripts with 3' termini in exon 1 in steady-state RNA from injected oocytes. Using a cell-free system to investigate the mechanism of premature 3' end formation, we found that polymerase II generates prominent ADA transcripts approximately 96 to 100 nucleotides in length which are similar to the major short transcripts found in steady-state RNA from oocytes injected with ADA templates. We have determined that these transcripts are the processed products of 108- to 112-nucleotide precursors. Precursor formation is (i) favored in reactions using circular templates, (ii) not the result of a posttranscriptional processing event, (iii) sensitive to low concentrations of Sarkosyl, and (iv) dependent on a factor(s) which is inactivated in crude extracts at 47 degrees C for 15 min. The cell-free system will allow further characterization of the template and factor requirements involved in the control of premature 3' end formation by RNA polymerase II.

scholarly journals December 2021 Pulmonary Case of the Month: Interstitial Lung Disease with Red Knuckles

2021 ◽  
Vol 23 (6) ◽  
pp. 151-154
Author(s):  
Richard Robbins ◽  
◽  
Stephen Klotz
Keyword(s):  
Interstitial Lung Disease ◽  
Inpatient Care ◽  
Rna Viruses ◽  
Specific Binding ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Dipeptidyl Peptidase 4 ◽  
A Cell ◽  
Coronavirus Infection

No abstract available. Article truncated after 150 words. We thought a follow-up to our original brief review of COVID-19 in February, 2020 might be useful. As we write this in early December 2021, we again caution that this area is rapidly changing and what is true today will likely be outdated tomorrow. We again borrowed heavily from the Centers for Disease Control (CDC) CDC website and the NIH website which have extensive discussions over numerous pages covering COVID-19. Our hope is to condense those recommendations. We do not discuss inpatient care in any detail. COVID-19 Variants The initial steps of coronavirus infection involve the specific binding of the coronavirus spike (S) protein to the cellular entry receptors which are normally on a cell. These include human aminopeptidase N (APN; HCoV-229E), angiotensin-converting enzyme 2 (ACE2; HCoV-NL63, SARS-CoV and SARS-CoV-2) and dipeptidyl peptidase 4 (DPP4; MERS-CoV). All viruses, but especially simple single-stranded RNA viruses like COVID-19, constantly change through mutation …

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