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 Functional TFIIH Is Required for UV-Induced Translocation of CSA to the Nuclear Matrix

2007 ◽  
Vol 27 (7) ◽  
pp. 2538-2547 ◽  
Author(s):  
Masafumi Saijo ◽  
Tamami Hirai ◽  
Akiko Ogawa ◽  
Aki Kobayashi ◽  
Shinya Kamiuchi ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Uv Irradiation ◽  
Nuclear Matrix ◽  
Mammalian Cells ◽  
Insoluble Fraction ◽  
Dnase I ◽  
Free System ◽  
Cell Free System ◽  
A Cell

ABSTRACT Transcription-coupled repair (TCR) efficiently removes a variety of lesions from the transcribed strand of active genes. Mutations in Cockayne syndrome group A and B genes (CSA and CSB) result in defective TCR, but the molecular mechanism of TCR in mammalian cells is not clear. We have found that CSA protein is translocated to the nuclear matrix after UV irradiation and colocalized with the hyperphosphorylated form of RNA polymerase II and that the translocation is dependent on CSB. We developed a cell-free system for the UV-induced translocation of CSA. A cytoskeleton (CSK) buffer-soluble fraction containing CSA and a CSK buffer-insoluble fraction prepared from UV-irradiated CS-A cells were mixed. After incubation, the insoluble fraction was treated with DNase I. CSA protein was detected in the DNase I-insoluble fraction, indicating that it was translocated to the nuclear matrix. In this cell-free system, the translocation was dependent on UV irradiation, CSB function, and TCR-competent CSA. Moreover, the translocation was dependent on functional TFIIH, as well as chromatin structure and transcription elongation. These results suggest that alterations of chromatin at the RNA polymerase II stall site, which depend on CSB and TFIIH at least, are necessary for the UV-induced translocation of CSA to the nuclear matrix.

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.

scholarly journals 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.

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.

Differential stability of c-myc mRNAS in a cell-free system

1988 ◽  
Vol 8 (7) ◽  
pp. 2860-2868
Author(s):  
R Pei ◽  
K Calame
Keyword(s):  
Full Length ◽  
Free System ◽  
In Vitro System ◽  
Rapid Degradation ◽  
Cell Free System ◽  
Exon 1 ◽  
A Cell ◽  
The Stability

We have developed a simple cell-free system for studying the stability of different mRNAs in vitro. We demonstrate that the threefold greater stability in vivo of truncated c-myc mRNA (lacking exon 1) compared with that of full-length c-myc mRNA is maintained in our in vitro system. Chimeric mRNAs in which the first exon of c-myc was fused to immunoglobulin C alpha heavy chain or glyceraldehyde-3-phosphate dehydrogenase mRNAs were not rapidly degraded, demonstrating that c-myc exon 1 alone is not sufficient to tag mRNAs for rapid degradation. Competition experiments show that full-length c-myc mRNA is specifically recognized by a factor(s) responsible for its rapid degradation. This system will allow further characterization and purification of these factors.

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.

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