Arabidopsis Pol II-Dependent in Vitro Transcription System Reveals Role of Chromatin for Light-Inducible rbcS Gene Transcription

ABSTRACT tagA, tagD, and tuaA operons are responsible for the synthesis of cell wall anionic polymer, teichoic acid, and teichuronic acid, respectively, in Bacillus subtilis. Under phosphate starvation conditions, teichuronic acid is synthesized while teichoic acid synthesis is inhibited. Expression of these genes is controlled by PhoP-PhoR, a two-component system. It has been proposed that PhoP∼P plays a key role in the activation oftuaA and the repression of tagA andtagD. In this study, we demonstrated the role of PhoP∼P in the switch process from teichoic acid synthesis to teichuronic acid synthesis, by using an in vitro transcription system. The results indicate that PhoP∼P is sufficient to repress the transcription of the tagA and tagD promoters and also to activate the transcription of the tuaA promoter.

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Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.1-15.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 1-15 ◽

Cited By ~ 25

Author(s):

Seiji Yamamoto ◽

Yoshinori Watanabe ◽

Peter J. van der Spek ◽

Tomomichi Watanabe ◽

Hiroyuki Fujimoto ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

In Vitro Transcription ◽

Pol Ii ◽

Transcription System ◽

Carboxy Terminal ◽

Transition To Elongation ◽

Ctd Phosphorylation

ABSTRACT The general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIEα and ceTFIIEβ). ceTFIIEβ could partially replace hTFIIEβ, whereas ceTFIIEα could not replace hTFIIEα. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIEα did not bind tightly to hTFIIEβ, and ceTFIIEβ showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIEβ induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIEβ was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.

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RPAP2 regulates a transcription initiation checkpoint by prohibiting assembly of preinitiation complex

10.1101/2021.06.18.448918 ◽

2021 ◽

Author(s):

Xizi Chen ◽

Yilun Qi ◽

Xinxin Wang ◽

Zhenning Wang ◽

Li Wang ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Phosphatase Activity ◽

Transcription Initiation ◽

Critical Role ◽

In Vitro Transcription ◽

Pol Ii ◽

Activity Structure

RNA polymerase II (Pol II)-mediated transcription in metazoan requires precise regulation. RNA polymerase II-associated protein 2 (RPAP2) was previously identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD). We found that RPAP2 binds hypo/hyper-phosphorylated Pol II with undetectable phosphatase activity. Structure of RPAP2-Pol II shows mutually exclusive assembly of RPAP2-Pol II and pre-initiation complex (PIC) due to three steric clashes. RPAP2 prevents/disrupts Pol II-TFIIF interaction and impairs in vitro transcription initiation, suggesting a function in prohibiting PIC assembly. Loss of RPAP2 in cells leads to global accumulation of TFIIF and Pol II at promoters, indicating critical role of RPAP2 in inhibiting PIC assembly independent of its putative phosphatase activity. Our study indicates that RPAP2 functions as a gatekeeper to prohibit PIC assembly and transcription initiation and suggests a novel transcription checkpoint.

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The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽

10.7554/elife.03573 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 21

Author(s):

Yick W Fong ◽

Jaclyn J Ho ◽

Carla Inouye ◽

Robert Tjian

Keyword(s):

Stem Cell ◽

Es Cells ◽

Embryonic Stem ◽

In Vitro Transcription ◽

Specific Gene ◽

Transcriptional Level ◽

Ips Cell ◽

Ribonucleoprotein Complex ◽

Transcription System

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.

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Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4799 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4799-4807 ◽

Cited By ~ 11

Author(s):

L J Brunet ◽

A J Berk

Keyword(s):

Adenovirus Type ◽

In Vitro Transcription ◽

Mrna Levels ◽

Adenovirus E1a ◽

Transcription System ◽

Nuclear Extracts ◽

Tumor Virus ◽

E1a Gene ◽

High Level

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.

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Fluorescent primer-based in vitro transcription system of viral RNA-dependent RNA polymerases

Analytical Biochemistry ◽

10.1016/j.ab.2012.10.025 ◽

2013 ◽

Vol 433 (2) ◽

pp. 92-94

Author(s):

Qiang Wang ◽

Leiyun Weng ◽

Hongbing Jiang ◽

Shijian Zhang ◽

Tetsuya Toyoda

Keyword(s):

In Vitro Transcription ◽

Rna Polymerases ◽

Viral Rna ◽

Transcription System

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Accurate transcription of a plant mitochondrial gene in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2035-2039.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2035-2039

Author(s):

P J Hanic-Joyce ◽

M W Gray

Keyword(s):

Mitochondrial Gene ◽

Plant Mitochondria ◽

In Vitro Transcription ◽

Dna Templates ◽

In Vitro System ◽

Transcription System ◽

Translation Initiation Codon ◽

Mitochondrial Extract

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.

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Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4799-4807.1988 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4799-4807

Author(s):

L J Brunet ◽

A J Berk

Keyword(s):

Adenovirus Type ◽

In Vitro Transcription ◽

Mrna Levels ◽

Adenovirus E1a ◽

Transcription System ◽

Nuclear Extracts ◽

Tumor Virus ◽

E1a Gene ◽

High Level

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.

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Regulation of protamine gene expression in an in vitro homologous system.

Acta Biochimica Polonica ◽

10.18388/abp.1996_4506 ◽

1996 ◽

Vol 43 (2) ◽

pp. 369-377 ◽

Cited By ~ 1

Author(s):

J M Jankowski ◽

P D Cannon ◽

F Van der Hoorn ◽

L D Wasilewska ◽

N C Wong ◽

...

Keyword(s):

Gene Expression ◽

Regulatory Elements ◽

In Vitro Transcription ◽

Tata Box ◽

Transcription System ◽

Proposed Model ◽

Binding Factor ◽

Gc Box ◽

Responsive Element

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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Differential role of cAMP, cGMP and Ca2+ and involvement of kinases and CBP-CREB-CRE pathway in regulation of arylalkylamine N-acetyltransferase 2 gene in the pineal organ of air-breathing catfish, Clarias gariepinus

10.21203/rs.3.rs-422474/v1 ◽

2021 ◽

Author(s):

Hijam Nonibala ◽

Braj Bansh Prasad Gupta

Keyword(s):

Gene Expression ◽

Map Kinase ◽

Gene Transcription ◽

Pineal Organ ◽

Clarias Gariepinus ◽

Specific Inhibitor ◽

P38 Map Kinase ◽

Camp And Cgmp

Abstract Transcription of arylalkylamine N-acetyltransferase 2 (aanat2) gene leads to formation of AANAT2 - the rate-limiting enzyme in melatonin synthesis pathway in photosensitive fish pineal organ. However, unlike in avian and mammalian pineal gland, there is practically no information on signal transduction pathway(s) involved in regulation of aanat2 gene transcription in the fish pineal organ. Therefore, we investigated the role of important molecular components of signalling via cAMP, cGMP, Ca2+ involving PKA, PKG, PKC, MeK and p38 MAP kinase as well as possible role of serine/threonine phosphatases, CREB and CBP using their specific inhibitors and/or activators in aanat2 gene transcription in the fish pineal organ maintained under in vitro culture-conditions. db-cAMP and db-cGMP stimulated the expression of aanat2 gene. db-cAMP- and cGMP-induced aanat2 gene expression was significantly reduced in the presence of H-89 (specific inhibitor of PKA), KT5823 (specific inhibitor of PKG), chelerythrine chloride (specific inhibitor of PKC), U0126 ethanolate (specific inhibitor of MeK) and SB 202190 monohydrochloride hydrate (specific inhibitor of p38 MAP kinase). Inhibitors of PP1 and PP2A significantly increased aanat2 gene expression as well as significantly reduced cAMP- and cGMP-induced gene transcription, while inhibitor of PP2B had no effect on aanat2 gene expression. Inhibitors of both CREB and CBP-CREB interaction completely blocked cAMP-induced aanat2 gene transcription. Based on these findings, we suggest that cAMP, cGMP and Ca2+ stimulate aanat2 gene transcription via PKA, PKG and PKC, respectively. Further, protein phosphatases and CBP-CREB-CRE pathway are actively involved in regulation of on aanat2 gene expression in the fish pineal organ.

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