scholarly journals Investigating transcription reinitiation through in vitro approaches

Transcription ◽  
2014 ◽  
Vol 5 (1) ◽  
pp. e27704 ◽  
Author(s):  
Giorgio Dieci ◽  
Beatrice Fermi ◽  
Maria Cristina Bosio
Keyword(s):  
Transcription Reinitiation

scholarly journals Mediator and p300/CBP-Steroid Receptor Coactivator Complexes Have Distinct Roles, but Function Synergistically, during Estrogen Receptor α-Dependent Transcription with Chromatin Templates

2003 ◽  
Vol 23 (1) ◽  
pp. 335-348 ◽  
Author(s):  
Mari Luz Acevedo ◽  
W. Lee Kraus
Keyword(s):  
Estrogen Receptor ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Preinitiation Complex ◽  
Transcription System ◽  
Steroid Receptor Coactivator ◽  
Transcription Reinitiation

ABSTRACT Ligand-dependent transcriptional activation by nuclear receptors involves the recruitment of various coactivators to the promoters of hormone-regulated genes assembled into chromatin. Nuclear receptor coactivators include histone acetyltransferase complexes, such as p300/CBP-steroid receptor coactivator (SRC), as well as the multisubunit mediator complexes (“Mediator”), which may help recruit RNA polymerase II to the promoter. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system, to examine the functional role for Mediator in the transcriptional activity of estrogen receptor α (ERα) with chromatin templates, as well as functional interplay between Mediator and p300/CBP during ERα-dependent transcription. Using three different approaches to functionally inactivate Mediator (immunoneutralization, immunodepletion, and inhibitory polypeptides), we find that Mediator is required for maximal transcriptional activation by ligand-activated ERα. In addition, we demonstrate synergism between Mediator and p300/CBP-SRC during ERα-dependent transcription with chromatin templates, but not with naked DNA. This synergism is important for promoting the formation of a stable transcription preinitiation complex leading to the initiation of transcription. Interestingly, we find that Mediator has an additional distinct role during ERα-dependent transcription not shared by p300/CBP-SRC: namely, to promote preinitiation complex formation for subsequent rounds of transcription reinitiation. These results suggest that one functional consequence of Mediator-ERα interactions is the stimulation of multiple cycles of transcription reinitiation. Collectively, our results indicate an important role for Mediator, as well as its functional interplay with p300/CBP-SRC, in the enhancement of ERα-dependent transcription with chromatin templates.

scholarly journals Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation

2017 ◽  
Author(s):  
Yoo Jin Joo ◽  
Scott B. Ficarro ◽  
Luis M. Soares ◽  
Yujin Chun ◽  
Jarrod A. Marto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Transcription Reinitiation ◽  
Promoter Dna ◽  
Basal Transcription Factors ◽  
Necessary And Sufficient

AbstractTFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-Binding Protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-Associated Factor (TAF) subunits recognize downstream promoter elements, act as co-activators, and interact with nucleosomes. Here we show that transcription induces stable TAF binding to downstream promoter DNA, independent of upstream contacts, TBP, or other basal transcription factors. This transcription-dependent TAF complex promotes subsequent activator-independent transcription, and promoter response to TAF mutations in vivo correlates with the level of downstream, rather than overall, Taf1 crosslinking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.

scholarly journals Transcription reinitiation rate: a special role for the TATA box.

1997 ◽  
Vol 17 (7) ◽  
pp. 3809-3816 ◽  
Author(s):  
D Yean ◽  
J Gralla
Keyword(s):  
Point Mutations ◽  
In Vitro Assays ◽  
Special Role ◽  
Promoter Elements ◽  
Tata Element ◽  
Transcription Reinitiation ◽  
Active Transcription ◽  
Transcription Complexes ◽  
Transcriptional Induction

Promoters need to specify both the timing of transcriptional induction and the amount of transcript synthesized. In order to explore each of these effects separately, in vitro assays for the level of active preinitiation complex formation and for the rate of continuous RNA production were done. The effects were found to be influenced differently by different promoter elements. A consensus TATA element had a very strong effect on the rate of continuous RNA production, whereas two types of activators were important primarily in forming active transcription preinitiation complexes. Consensus TATA promoters exhibited high rates of continuous transcription; they assembled active preinitiation transcription complexes slowly but then produced transcripts continuously at an approximately fivefold-higher rate. Initiator-containing TATA-less promoters produced continuous transcripts slowly. Point mutations in the TATA element led to lower levels of transcription by reducing the number of preinitiation complexes and amplifying this reduction by lowering the apparent reinitiation rate. The results allow understanding of the sequence diversity of promoter elements in terms of specifying separate controls over the sensitivity of gene induction and over the strength of the induced promoter.

scholarly journals Activator-Independent Functions of the Yeast Mediator Sin4 Complex in Preinitiation Complex Formation and Transcription Reinitiation

2003 ◽  
Vol 23 (1) ◽  
pp. 349-358 ◽  
Author(s):  
Wendy M. Reeves ◽  
Steven Hahn
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Preinitiation Complex ◽  
In Vitro System ◽  
Pol Ii ◽  
Independent Functions ◽  
Transcription Reinitiation ◽  
Reduced Rate

ABSTRACT RNA polymerase II (Pol II) Mediator plays an essential role in both basal and activated transcription. Previously, subunits of the Sin4 Mediator complex (Sin4, Pgd1, Gal11, and Med2) have been implicated in both positive and negative transcriptional regulation. Furthermore, it was proposed that this subcomplex constitutes an activator-binding domain. A yeast nuclear-extract system was used to investigate the biochemical role of the Sin4 complex. In contrast to previous findings, we found at least two general activator-independent roles for the Sin4 complex. First, mutations in sin4 and pgd1 destabilized the Pol II-Med complex, leading to a reduced rate and extent of preinitiation complex (PIC) formation both in the presence and absence of activators. Although reduced in amount compared with the wild type, PICs that are formed lacking the Sin4 complex are stable and can initiate transcription normally. Second, mutation of pgd1 causes partial disruption of the Sin4 complex and leads to a defect in transcription reinitiation. This defect is caused by dissociation of mutant Mediator from promoters after initiation, leading to nonfunctional Scaffold complexes. These results show that function of the Sin4 complex is not essential for transcription activation in a crude in vitro system but that it plays key roles in the general transcription mechanism.

scholarly journals The transcription reinitiation properties of RNA polymerase III in the absence of transcription factors

2008 ◽  
Vol 13 (1) ◽  
Author(s):  
Roberto Ferrari ◽  
Giorgio Dieci
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Transcription Unit ◽  
Transcription System ◽  
Transcription Reinitiation ◽  
Pol Iii

AbstractTranscription reinitiation by RNA polymerase (Pol) III proceeds through facilitated recycling, a process by which the terminating Pol III, assisted by the transcription factors TFIIIB and TFIIIC, rapidly reloads onto the same transcription unit. To get further insight into the Pol III transcription mechanism, we analyzed the kinetics of transcription initiation and reinitiation of a simplified in vitro transcription system consisting only of Pol III and template DNA. The data indicates that, in the absence of transcription factors, first-round transcription initiation by Pol III proceeds at a normal rate, while facilitated reinitiation during subsequent cycles is compromised.

Ultrastructural Investigations of the Contractile Filaments of Amoebae

1974 ◽  
Vol 32 ◽  
pp. 188-189
Author(s):  
P.L. Moore
Keyword(s):  
Cytoplasmic Streaming ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Amoeboid Movement ◽  
Different Types ◽  
Chemical Conditions ◽  
Complete Interpretation

Previous freeze fracture results on the intact giant, amoeba Chaos carolinensis indicated the presence of a fibrillar arrangement of filaments within the cytoplasm. A complete interpretation of the three dimensional ultrastructure of these structures, and their possible role in amoeboid movement was not possible, since comparable results could not be obtained with conventional fixation of intact amoebae. Progress in interpreting the freeze fracture images of amoebae required a more thorough understanding of the different types of filaments present in amoebae, and of the ways in which they could be organized while remaining functional.The recent development of a calcium sensitive, demembranated, amoeboid model of Chaos carolinensis has made it possible to achieve a better understanding of such functional arrangements of amoeboid filaments. In these models the motility of demembranated cytoplasm can be controlled in vitro, and the chemical conditions necessary for contractility, and cytoplasmic streaming can be investigated. It is clear from these studies that “fibrils” exist in amoeboid models, and that they are capable of contracting along their length under conditions similar to those which cause contraction in vertebrate muscles.

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

Induction and Differentiation of Dental Epithelium in Vivo and in Vitro

1982 ◽  
Vol 40 ◽  
pp. 142-145
Author(s):  
E. J. Kollar
Keyword(s):  
Connective Tissue ◽  
Oral Mucosa ◽  
Basal Lamina ◽  
Functional Derivatives ◽  
Specific Source ◽  
Dental Epithelium ◽  
Connective Tissue Stroma

The differentiation and maintenance of many specialized epithelial structures are dependent on the underlying connective tissue stroma and on an intact basal lamina. These requirements are especially stringent in the development and maintenance of the skin and oral mucosa. The keratinization patterns of thin or thick cornified layers as well as the appearance of specialized functional derivatives such as hair and teeth can be correlated with the specific source of stroma which supports these differentiated expressions.

Immunoenzymatic demonstration of the simultaneous presence of transferrin, hemopexin and albumin in a same hepatocyte and their ultrastructural localization

1978 ◽  
Vol 36 (2) ◽  
pp. 88-89
Author(s):  
M. Kraemer ◽  
J. Foucrier ◽  
J. Vassy ◽  
M.T. Chalumeau
Keyword(s):  
Plasma Proteins ◽  
Rat Hepatocytes ◽  
Ultrastructural Localization ◽  
Carrier Proteins ◽  
Normal Cells ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Monospecific Antisera ◽  
Different Levels

Some authors using immunofluorescent techniques had already suggested that some hepatocytes are able to synthetize several plasma proteins. In vitro studies on normal cells or on cells issued of murine hepatomas raise the same conclusion. These works could be indications of an hepatocyte functionnal non-specialization, meanwhile the authors never give direct topographic proofs suitable with this hypothesis.The use of immunoenzymatic techniques after obtention of monospecific antisera had seemed to us useful to bring forward a better knowledge of this problem. We have studied three carrier proteins (transferrin = Tf, hemopexin = Hx, albumin = Alb) operating at different levels in iron metabolism by demonstrating and localizing the adult rat hepatocytes involved in their synthesis.Immunological, histological and ultrastructural methods have been described in a previous work.

Sign in / Sign up

Export Citation Format

Share Document