scholarly journals Identification and characterization of transcriptional arrest sites in exon 1 of the human adenosine deaminase gene.

1990 ◽  
Vol 10 (9) ◽  
pp. 4555-4564 ◽  
Author(s):  
Z Chen ◽  
M L Harless ◽  
D A Wright ◽  
R E Kellems
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Adenosine Deaminase ◽  
System Analysis ◽  
Proximal Region ◽  
Xenopus Laevis Oocyte ◽  
Transcription Analysis ◽  
Transcription System ◽  
Exon 1 ◽  
Promoter Proximal Region

Analysis of human adenosine deaminase (ADA) gene transcription in four different cell lines indicated that a high density of RNA polymerase II complexes is present at the 5' end of the gene and that the extent of transcription elongation beyond the promoter-proximal region governs gene expression. To determine the sequence requirements for a potential transcription arrest site in the promoter-proximal region, genomic clones containing the ADA promoter, exon 1, and various lengths of intron 1 were injected into Xenopus laevis oocyte germinal vesicles. Transcription analysis indicated that nascent ADA transcripts were highly represented at the promoter-proximal region of the injected templates, suggesting that transcription arrest occurred in the oocyte transcription system. Analysis of the transcription products indicated that ADA transcription initiated at the authentic start site and that the most prominent, short ADA transcripts were 105 nucleotides in length. The 3' end of these transcripts mapped within exon 1, 10 nucleotides downstream of the translation initiation codon. Deletion analysis demonstrated that sequences within exon 1 were sufficient to specify the synthesis of the 105-nucleotide transcripts. Taken together, these data suggest that a transcription arrest mechanism operates in the promoter-proximal region of the human ADA gene and that regulation of elongation beyond this point plays a major role in regulating ADA gene expression.

Identification and characterization of transcriptional arrest sites in exon 1 of the human adenosine deaminase gene

1990 ◽  
Vol 10 (9) ◽  
pp. 4555-4564
Author(s):  
Z Chen ◽  
M L Harless ◽  
D A Wright ◽  
R E Kellems
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Adenosine Deaminase ◽  
System Analysis ◽  
Proximal Region ◽  
Xenopus Laevis Oocyte ◽  
Transcription Analysis ◽  
Transcription System ◽  
Exon 1 ◽  
Promoter Proximal Region

Analysis of human adenosine deaminase (ADA) gene transcription in four different cell lines indicated that a high density of RNA polymerase II complexes is present at the 5' end of the gene and that the extent of transcription elongation beyond the promoter-proximal region governs gene expression. To determine the sequence requirements for a potential transcription arrest site in the promoter-proximal region, genomic clones containing the ADA promoter, exon 1, and various lengths of intron 1 were injected into Xenopus laevis oocyte germinal vesicles. Transcription analysis indicated that nascent ADA transcripts were highly represented at the promoter-proximal region of the injected templates, suggesting that transcription arrest occurred in the oocyte transcription system. Analysis of the transcription products indicated that ADA transcription initiated at the authentic start site and that the most prominent, short ADA transcripts were 105 nucleotides in length. The 3' end of these transcripts mapped within exon 1, 10 nucleotides downstream of the translation initiation codon. Deletion analysis demonstrated that sequences within exon 1 were sufficient to specify the synthesis of the 105-nucleotide transcripts. Taken together, these data suggest that a transcription arrest mechanism operates in the promoter-proximal region of the human ADA gene and that regulation of elongation beyond this point plays a major role in regulating ADA gene expression.

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 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 Transcriptional Pausing Caused by NELF Plays a Dual Role in Regulating Immediate-Early Expression of the junB Gene

2006 ◽  
Vol 26 (16) ◽  
pp. 6094-6104 ◽  
Author(s):  
Masatoshi Aida ◽  
Yexi Chen ◽  
Koichi Nakajima ◽  
Yuki Yamaguchi ◽  
Tadashi Wada ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Mrna Level ◽  
Proximal Region ◽  
Negative Regulation ◽  
Immediate Early ◽  
Dual Function ◽  
Early Expression ◽  
Transcriptional Pausing ◽  
Promoter Proximal Region

ABSTRACT Human 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) negatively regulate transcription elongation by RNA polymerase II (RNAPII) in vitro. However, the physiological roles of this negative regulation are not well understood. Here, by using a number of approaches to identify protein-DNA interactions in vivo, we show that DSIF- and NELF-mediated transcriptional pausing has a dual function in regulating immediate-early expression of the human junB gene. Before induction by interleukin-6, RNAPII, DSIF, and NELF accumulate in the promoter-proximal region of junB, mainly at around position +50 from the transcription initiation site. After induction, the association of these proteins with the promoter-proximal region continues whereas RNAPII and DSIF are also found in the downstream regions. Depletion of a subunit of NELF by RNA interference enhances the junB mRNA level both before and after induction, indicating that DSIF- and NELF-mediated pausing contributes to the negative regulation of junB expression, not only by inducing RNAPII pausing before induction but also by attenuating transcription after induction. These regulatory mechanisms appear to be conserved in other immediate-early genes as well.

scholarly journals RNA Polymerase II Accumulation in the Promoter-Proximal Region of the Dihydrofolate Reductase and γ-Actin Genes

2003 ◽  
Vol 23 (6) ◽  
pp. 1961-1967 ◽  
Author(s):  
Chonghui Cheng ◽  
Phillip A. Sharp
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dihydrofolate Reductase ◽  
Mammalian Cells ◽  
Proximal Region ◽  
Heptad Repeat ◽  
Pol Ii ◽  
Actin Genes ◽  
Carboxyl Terminal ◽  
Promoter Proximal Region

ABSTRACT The carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) can be phosphorylated at serine 2 (Ser-2) and serine 5 (Ser-5) of the CTD heptad repeat YSPTSPS, and this phosphorylation is important in coupling transcription to RNA processing, including 5′ capping, splicing, and polyadenylation. The mammalian endogenous dihydrofolate reductase and γ-actin genes have been used to study the association of Pol II with different regions of transcribed genes (promoter-proximal compared to distal regions) and the phosphorylation status of its CTD. For both genes, Pol II is more concentrated in the promoter-proximal regions than in the interior regions. Moreover, different phosphorylation forms of Pol II are associated with distinct regions. Ser-5 phosphorylation of Pol II is concentrated near the promoter, while Ser-2 phosphorylation is observed throughout the gene. These results suggest that the accumulation of paused Pol II in promoter-proximal regions may be a common feature of gene regulation in mammalian cells.

scholarly journals Structure of transcribed chromatin is a sensor of DNA damage

2015 ◽  
Vol 1 (6) ◽  
pp. e1500021 ◽  
Author(s):  
Nikolay A. Pestov ◽  
Nadezhda S. Gerasimova ◽  
Olga I. Kulaeva ◽  
Vasily M. Studitsky
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Supercoiling ◽  
Proximal Region ◽  
Cellular Systems ◽  
Dna Breaks ◽  
Specific Mechanism ◽  
Nucleosome Structure ◽  
Nucleosomal Dna ◽  
Promoter Proximal Region

Early detection and repair of damaged DNA is essential for cell functioning and survival. Although multiple cellular systems are involved in the repair of single-strand DNA breaks (SSBs), it remains unknown how SSBs present in the nontemplate strand (NT-SSBs) of DNA organized in chromatin are detected. The effect of NT-SSBs on transcription through chromatin by RNA polymerase II was studied. NT-SSBs localized in the promoter-proximal region of nucleosomal DNA and hidden in the nucleosome structure can induce a nearly quantitative arrest of RNA polymerase downstream of the break, whereas more promoter-distal SSBs moderately facilitate transcription. The location of the arrest sites on nucleosomal DNA suggests that formation of small intranucleosomal DNA loops causes the arrest. This mechanism likely involves relief of unconstrained DNA supercoiling accumulated during transcription through chromatin by NT-SSBs. These data suggest the existence of a novel chromatin-specific mechanism that allows the detection of NT-SSBs by the transcribing enzyme.

scholarly journals CDK9-dependent RNA polymerase II pausing controls transcription initiation

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Saskia Gressel ◽  
Björn Schwalb ◽  
Tim Michael Decker ◽  
Weihua Qin ◽  
Heinrich Leonhardt ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Human Cell Line ◽  
Pause Duration ◽  
Proximal Region ◽  
Chromatin Interactions ◽  
Initiation Frequency ◽  
Polymerase Pausing ◽  
Promoter Proximal Region

Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a ‘multi-omics’ approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells (‘pause-initiation limit’). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.

scholarly journals Sequence requirements for transcriptional arrest in exon 1 of the human adenosine deaminase gene.

1991 ◽  
Vol 11 (12) ◽  
pp. 6248-6256 ◽  
Author(s):  
Z Chen ◽  
J W Innis ◽  
M H Sun ◽  
D A Wright ◽  
R E Kellems
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Adenosine Deaminase ◽  
Rna Polymerase Iii ◽  
Independent Manner ◽  
Specific Expression ◽  
Naturally Occurring ◽  
Exon 1 ◽  
Dna Fragment ◽  
Sequence Requirements

We have previously demonstrated that a transcriptional arrest site exists in exon 1 of the human adenosine deaminase (ADA) gene and that this site may play a role in ADA gene expression (Z. Chen, M. L. Harless, D. A. Wright, and R. E. Kellems, Mol. Cell. Biol. 10:4555-4564, 1990). Sequences involved in this process are not known precisely. To further define the template requirements for transcriptional arrest within exon 1 of the human ADA gene, various ADA templates were constructed and their abilities to confer transcriptional arrest were determined following injection into Xenopus oocytes. The exon 1 transcriptional arrest signal functioned downstream of several RNA polymerase II promoters and an RNA polymerase III promoter, implying that the transcriptional arrest site in exon 1 of the ADA gene is promoter independent. We identified a 43-bp DNA fragment which functions as a transcriptional arrest signal. Additional studies showed that the transcriptional arrest site functioned only in the naturally occurring orientation. Therefore, we have identified a 43-bp DNA fragment which functions as a transcriptional arrest signal in an orientation-dependent and promoter-independent manner. On the basis of our findings, we hypothesize that tissue-specific expression of the ADA gene is governed by factors that function as antiterminators to promote transcriptional readthrough of the exon 1 transcriptional arrest site.

Sequence requirements for transcriptional arrest in exon 1 of the human adenosine deaminase gene

1991 ◽  
Vol 11 (12) ◽  
pp. 6248-6256
Author(s):  
Z Chen ◽  
J W Innis ◽  
M H Sun ◽  
D A Wright ◽  
R E Kellems
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Adenosine Deaminase ◽  
Rna Polymerase Iii ◽  
Independent Manner ◽  
Specific Expression ◽  
Naturally Occurring ◽  
Exon 1 ◽  
Dna Fragment ◽  
Sequence Requirements

We have previously demonstrated that a transcriptional arrest site exists in exon 1 of the human adenosine deaminase (ADA) gene and that this site may play a role in ADA gene expression (Z. Chen, M. L. Harless, D. A. Wright, and R. E. Kellems, Mol. Cell. Biol. 10:4555-4564, 1990). Sequences involved in this process are not known precisely. To further define the template requirements for transcriptional arrest within exon 1 of the human ADA gene, various ADA templates were constructed and their abilities to confer transcriptional arrest were determined following injection into Xenopus oocytes. The exon 1 transcriptional arrest signal functioned downstream of several RNA polymerase II promoters and an RNA polymerase III promoter, implying that the transcriptional arrest site in exon 1 of the ADA gene is promoter independent. We identified a 43-bp DNA fragment which functions as a transcriptional arrest signal. Additional studies showed that the transcriptional arrest site functioned only in the naturally occurring orientation. Therefore, we have identified a 43-bp DNA fragment which functions as a transcriptional arrest signal in an orientation-dependent and promoter-independent manner. On the basis of our findings, we hypothesize that tissue-specific expression of the ADA gene is governed by factors that function as antiterminators to promote transcriptional readthrough of the exon 1 transcriptional arrest site.

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