Characterization of a mammalian ribosomal protein gene promoter

1990 ◽  
Vol 68 (6) ◽  
pp. 949-956 ◽  
Author(s):  
Peter Zahradka ◽  
Dawn E. Larson ◽  
Bruce H. Sells
Keyword(s):  
Ribosomal Protein ◽  
Rna Polymerase Ii ◽  
Dihydrofolate Reductase ◽  
Protein Gene ◽  
Gene Promoter ◽  
Ribosomal Protein Gene ◽  
Late Promoter ◽  
Promoter Elements ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter

The presence of specific promoter elements, notably the TATA and GC boxes, has been useful for categorizing genes transcribed by RNA polymerase II. The gene for the murine ribosomal protein (r-protein) L32 lacks both of these elements, although it has GC-rich regions. The conditions required for its optimal synthesis in vitro, however, resemble the properties of promoters containing TATA (adenovirus major late promoter) rather than GC boxes (dihydrofolate reductase). To further investigate the relationship of the r-protein gene to different promoter elements, transcription competition analyses were used to distinguish the presence of common protein-binding sequences. The low levels of competition observed by either the adenovirus major late promoter or dihydrofolate reductase promoter with the r-protein gene promoter resulted from general transcription factors present in each initiation complex. Competition by factors binding to common sequence elements was not observed, indicating the r-protein L32 gene possesses elements distinct from those present in the other genes examined.Key words: ribosomal protein gene, gene promoter, cell-free transcription.

scholarly journals Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II.

1991 ◽  
Vol 11 (3) ◽  
pp. 1195-1206 ◽  
Author(s):  
E Bengal ◽  
O Flores ◽  
A Krauskopf ◽  
D Reinberg ◽  
Y Aloni
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gel Filtration ◽  
Late Promoter ◽  
Elongation Complex ◽  
Cell Extracts ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter ◽  
Transcription Complexes

We have used a recently developed system that allows the isolation of complexes competent for RNA polymerase II elongation (E. Bengal, A. Goldring, and Y. Aloni, J. Biol. Chem. 264:18926-18932, 1989). Pulse-labeled transcription complexes were formed at the adenovirus major late promoter with use of HeLa cell extracts. Elongation-competent complexes were purified from most of the proteins present in the extract, as well as from loosely bound elongation factors, by high-salt gel filtration chromatography. We found that under these conditions the nascent RNA was displaced from the DNA during elongation. These column-purified complexes were used to analyze the activities of different transcription factors during elongation by RNA polymerase II. We found that transcription factor IIS (TFIIS), TFIIF, and TFIIX affected the efficiency of elongation through the adenovirus major late promoter attenuation site and a synthetic attenuation site composed of eight T residues. These factors have distinct activities that depend on whether they are added before RNA polymerase has reached the attenuation site or at the time when the polymerase is pausing at the attenuation site. TFIIS was found to have antiattenuation activity, while TFIIF and TFIIX stimulated the rate of elongation. In comparison with TFIIF, TFIIS is loosely bound to the elongation complex. We also found that the activities of the factors are dependent on the nature of the attenuator. These results indicate that at least three factors play a major role during elongation by RNA polymerase II.

Ribosomal protein gene promoter-based system for selective assessment of post-transcriptional regulation of cytokine genes

Cytokine ◽  
2009 ◽  
Vol 48 (1-2) ◽  
pp. 124-125
Author(s):  
Edward Hitti ◽  
Suhad Al-Yahya ◽  
Maher Al-Saif ◽  
Peer Mohideen ◽  
Lina Omar ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Ribosomal Protein ◽  
Protein Gene ◽  
Gene Promoter ◽  
Ribosomal Protein Gene ◽  
Cytokine Genes ◽  
Post Transcriptional Regulation

scholarly journals Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression.

1982 ◽  
Vol 2 (11) ◽  
pp. 1304-1319 ◽  
Author(s):  
R J Kaufman ◽  
P A Sharp
Keyword(s):  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Transcription Initiation ◽  
Simian Virus 40 ◽  
Variable Region ◽  
Polyadenylation Signal ◽  
Immunoglobulin Gene ◽  
Late Promoter ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter

Dihydrofolate reductase (DHFR) modular genes have been constructed with segments containing the adenovirus major late promoter, a 3' splice site from a variable region immunoglobulin gene, a DHFR cDNA, and portions of the simian virus 40 (SV40) genome. DNA-mediated transfer of these genes transformed Chinese hamster ovary DHFR- cells to the DHFR+ phenotype. Transformants contained one to several copies of the transfected DNA integrated into the host genome. Clones subjected to growth in increasing concentrations of methotrexate eventually gave rise to lines containing several hundred copies of the transforming DNA. Analysis of the DHFR mRNA produced in amplified lines indicated the following. (i) All clones utilize the adenovirus major late promoter for transcription initiation. (ii) A hybrid intron formed by the 5' splice site of the adenovirus major late leader and a 3' splice site from a variable-region immunoglobulin gene is properly excised. (iii) The mRNA is not efficiently polyadenylated at sequences in the 3' end of the DHFR cDNA but rather uses polyadenylation signals downstream from the DHFR cDNA. Three independent clones produce a DHFR mRNA containing SV40 or pBR322 and SV40 sequences, and the RNA is polyadenylated at the SV40 late polyadenylation site. Another clone has recombined into cellular DNA and apparently uses a cellular sequence for polyadenylation. Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DHFR cDNA gene generated a recombinant capable of transforming cells to the DHFR+ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenylation signal. Attachment of a DNA segment containing the transcription enhancer (72-base pair repeat) of SV40 further increased the biological activity of the modular DHFR gene 50- to 100-fold.

Factors involved in specific transcription by mammalian RNA polymerase II: role of transcription factors IIA, IID, and IIB during formation of a transcription-competent complex

1990 ◽  
Vol 10 (12) ◽  
pp. 6335-6347
Author(s):  
E Maldonado ◽  
I Ha ◽  
P Cortes ◽  
L Weis ◽  
D Reinberg
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Transcriptional Start Site ◽  
Late Promoter ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter ◽  
Tata Motif ◽  
Single Polypeptide ◽  
The Stability

Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.

scholarly journals Factors involved in specific transcription by mammalian RNA polymerase II: role of transcription factors IIA, IID, and IIB during formation of a transcription-competent complex.

1990 ◽  
Vol 10 (12) ◽  
pp. 6335-6347 ◽  
Author(s):  
E Maldonado ◽  
I Ha ◽  
P Cortes ◽  
L Weis ◽  
D Reinberg
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Transcriptional Start Site ◽  
Late Promoter ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter ◽  
Tata Motif ◽  
Single Polypeptide ◽  
The Stability

Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.

Promoter elements controlling developmental and environmental regulation of a tobacco ribosomal protein gene L34

1996 ◽  
Vol 32 (6) ◽  
pp. 1055-1065 ◽  
Author(s):  
Ziyu Dai ◽  
Jianwei Gao ◽  
Kyungsook An ◽  
James M. Lee ◽  
Gerald E. Edwards ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Environmental Regulation ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Promoter Elements

Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression

1982 ◽  
Vol 2 (11) ◽  
pp. 1304-1319
Author(s):  
R J Kaufman ◽  
P A Sharp
Keyword(s):  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Transcription Initiation ◽  
Simian Virus 40 ◽  
Variable Region ◽  
Polyadenylation Signal ◽  
Immunoglobulin Gene ◽  
Late Promoter ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter

Dihydrofolate reductase (DHFR) modular genes have been constructed with segments containing the adenovirus major late promoter, a 3' splice site from a variable region immunoglobulin gene, a DHFR cDNA, and portions of the simian virus 40 (SV40) genome. DNA-mediated transfer of these genes transformed Chinese hamster ovary DHFR- cells to the DHFR+ phenotype. Transformants contained one to several copies of the transfected DNA integrated into the host genome. Clones subjected to growth in increasing concentrations of methotrexate eventually gave rise to lines containing several hundred copies of the transforming DNA. Analysis of the DHFR mRNA produced in amplified lines indicated the following. (i) All clones utilize the adenovirus major late promoter for transcription initiation. (ii) A hybrid intron formed by the 5' splice site of the adenovirus major late leader and a 3' splice site from a variable-region immunoglobulin gene is properly excised. (iii) The mRNA is not efficiently polyadenylated at sequences in the 3' end of the DHFR cDNA but rather uses polyadenylation signals downstream from the DHFR cDNA. Three independent clones produce a DHFR mRNA containing SV40 or pBR322 and SV40 sequences, and the RNA is polyadenylated at the SV40 late polyadenylation site. Another clone has recombined into cellular DNA and apparently uses a cellular sequence for polyadenylation. Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DHFR cDNA gene generated a recombinant capable of transforming cells to the DHFR+ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenylation signal. Attachment of a DNA segment containing the transcription enhancer (72-base pair repeat) of SV40 further increased the biological activity of the modular DHFR gene 50- to 100-fold.

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