scholarly journals Development of an efficient in vitro transcription system for bloodstream form Trypanosoma brucei reveals life cycle-independent functionality of class I transcription factor A

2012 ◽  
Vol 181 (1) ◽  
pp. 29-36 ◽  
Author(s):  
Sung Hee Park ◽  
Tu N. Nguyen ◽  
Arthur Günzl
2021 ◽  
Author(s):  
Julia L Daiß ◽  
Michael Pilsl ◽  
Kristina Straub ◽  
Andrea Bleckmann ◽  
Mona Höcherl ◽  
...  

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP-fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG-box incapable of DNA-binding which may serve as a downstream-transcription factor binding platform in metazoans. Biochemical analysis and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG-box domain containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.


2001 ◽  
Vol 21 (15) ◽  
pp. 5223-5231 ◽  
Author(s):  
Sofia Georgieva ◽  
Elena Nabirochkina ◽  
F. Jeffrey Dilworth ◽  
Holger Eickhoff ◽  
Peter Becker ◽  
...  

ABSTRACT Weak hypomorph mutations in the enhancer of yellowgenes, e(y)1 and e(y)2, of Drosophila melanogaster were discovered during the search for genes involved in the organization of interaction between enhancers and promoters. Previously, the e(y)1 gene was cloned and found to encode TAFII40 protein. Here we cloned the e(y)2 gene and demonstrated that it encoded a new ubiquitous evolutionarily conserved transcription factor. The e(y)2 gene is located at 10C3 (36.67) region and is expressed at all stages ofDrosophila development. It encodes a 101-amino-acid protein, e(y)2. Vertebrates, insects, protozoa, and plants have proteins which demonstrate a high degree of homology to e(y)2. The e(y)2 protein is localized exclusively to the nuclei and is associated with numerous sites along the entire length of the salivary gland polytene chromosomes. Both genetic and biochemical experiments demonstrate an interaction between e(y)2 and TAFII40, while immunoprecipitation studies demonstrate that the major complex, including both proteins, appears to be distinct from TFIID. Furthermore, we provide genetic evidence suggesting that the carboxy terminus of dTAFII40 is important for mediating this interaction. Finally, using an in vitro transcription system, we demonstrate that recombinant e(y)2 is able to enhance transactivation by GAL4-VP16 on chromatin but not on naked DNA templates, suggesting that this novel protein is involved in the regulation of transcription.


2005 ◽  
Vol 83 (2) ◽  
pp. 188-195 ◽  
Author(s):  
Chenchen Lin ◽  
Meiyao Lin ◽  
Hungwen Chen

Glial cells missing (GCM) proteins are a novel family of zinc-containing transcription factors. Human GCMa/1 is primarily expressed in placental trophoblast cells and regulates SYNCYTIN gene expression, which mediates fusion of cytotrophoblasts to form the syncytiotrophoblast layer of the human placenta. To biochemically characterize the transcriptional activity of GCMa/1, we set up an in vitro transcription system for human GCMa/1 (hGCMa/1). Using G-free reporter constructs carrying multiple copies of wild-type or mutant GCMa-binding site (GBS) in front of a synthetic TATA box, we observed specific transcriptional activities of recombinant hGCMa/1 proteins prepared from a baculovirus – insect cell or Escherichia coli expression system. We further characterized GCMa/1-mediated tran scriptional activation on the native syncytin promoter. Using G-free reporter constructs containing the native syncytin promoter, a TATA box downstream of the proximal GBS in the syncytin promoter was shown to be essential for the transcription activation directed by hGCMa/1. Therefore, our results demonstrate positive transcriptional activities of GCMa/1 in vitro and provide a better understanding of GCMa/1-mediated SYNCYTIN gene expression.Key words: syncytin, transcription factor, GCMa/1, placenta.


eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.


1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807 ◽  
Author(s):  
L J Brunet ◽  
A J Berk

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.


2013 ◽  
Vol 433 (2) ◽  
pp. 92-94
Author(s):  
Qiang Wang ◽  
Leiyun Weng ◽  
Hongbing Jiang ◽  
Shijian Zhang ◽  
Tetsuya Toyoda

1991 ◽  
Vol 11 (4) ◽  
pp. 2035-2039
Author(s):  
P J Hanic-Joyce ◽  
M W Gray

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.


1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807
Author(s):  
L J Brunet ◽  
A J Berk

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.


1996 ◽  
Vol 43 (2) ◽  
pp. 369-377 ◽  
Author(s):  
J M Jankowski ◽  
P D Cannon ◽  
F Van der Hoorn ◽  
L D Wasilewska ◽  
N C Wong ◽  
...  

An in vitro transcription system from the trout testis nuclei was developed to study trout protamine gene expression. The protamine promoter contains, among others, two regulatory elements: 1) a cAMP-responsive element or CRE element (TGACGTCA) which is present in position 5' to TATA box, and 2) GC box (CCGCCC) which is present in position 3' to TATA box. The removal of the CRE-binding protein by titration (by the addition of appropriate oligonucleotides to the incubation mixture) resulted in a decrease in transcription of the protamine gene. These results were confirmed by experiments in which the pure CRE-binding factor (TPBP1) was used, as well as by those where a stimulatory effect of cAMP on protamine promoter transcription was observed. On the other hand, addition of oligonucleotides containing the GC-box sequence enhanced the protamine gene transcription indicating that the protein (Sp1 like) which binds to this sequence acts as a repressor of protamine gene expression. These results confirm the previously proposed model which suggested that the GC box played a role in negative regulation of the protamine gene expression. Involvement of some other factors in this process was also discussed.


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