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1994
Meissner W, Wanandi I, Carbon P, Krol A, Seifart K H
Transcription factors required for the expression of Xenopus laevis selenocysteine tRNA in vitro Article de journal
Dans: Nucleic Acids Res, vol. 22, no. 4, p. 553-559, 1994, ISBN: 8127703, (0305-1048 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Amino Acid-Specific/*genetics Support, Animals Base Sequence DNA-Binding Proteins/*physiology Human Molecular Sequence Data Oligonucleotide Probes RNA, Genetic Xenopus laevis, Non-U.S. Gov't TATA Box Transcription Factors/*genetics Transcription, Transfer, Unité ARN
@article{,
title = {Transcription factors required for the expression of Xenopus laevis selenocysteine tRNA in vitro},
author = {W Meissner and I Wanandi and P Carbon and A Krol and K H Seifart},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8127703},
isbn = {8127703},
year = {1994},
date = {1994-01-01},
journal = {Nucleic Acids Res},
volume = {22},
number = {4},
pages = {553-559},
abstract = {It has previously been reported that transcription in vivo of the tRNA(Sec) gene requires three promoter elements, a PSE and a TATA-box upstream of the coding region which are functionally interchangeable with the U6 snRNA gene counterparts and an internal B-block, resembling that of classical tRNA genes (1). We have established an in vitro transcription system from HeLa cells in which three factors, which are either essential for or stimulate transcription were identified. Apart from the TATA-binding protein TBP, the PSE-binding protein PBP was found to be essentially required for expression of the gene. Depletion of PBP from cell extracts by PSE-oligonucleotides abolished tRNA(Sec) transcription, which could be reconstituted by readdition of partially purified PBP. Addition of increasing amounts of recombinant human TBP to an S100 extract stimulated transcription of the tRNA(Sec), the mouse U6 snRNA and the human Y3 genes, an effect which was not observed in the case of a TATA-less tRNA gene. Purified human TFIIA strongly stimulated tRNA(Sec) transcription in a fashion depending on the concentration of TBP. Surprisingly, partially purified TFIIIC was shown to be dispensable for transcription in vitro and unable to bind the B-block of this gene in vitro, although its sequence matches the consensus for this element. Collectively, these data suggest that the mechanism by which transcription complexes are formed on the tRNA(Sec) gene is dramatically different from that observed for classical tRNA genes and much more resembles that observed for externally controlled pol III genes.},
note = {0305-1048
Journal Article},
keywords = {Amino Acid-Specific/*genetics Support, Animals Base Sequence DNA-Binding Proteins/*physiology Human Molecular Sequence Data Oligonucleotide Probes RNA, Genetic Xenopus laevis, Non-U.S. Gov't TATA Box Transcription Factors/*genetics Transcription, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
It has previously been reported that transcription in vivo of the tRNA(Sec) gene requires three promoter elements, a PSE and a TATA-box upstream of the coding region which are functionally interchangeable with the U6 snRNA gene counterparts and an internal B-block, resembling that of classical tRNA genes (1). We have established an in vitro transcription system from HeLa cells in which three factors, which are either essential for or stimulate transcription were identified. Apart from the TATA-binding protein TBP, the PSE-binding protein PBP was found to be essentially required for expression of the gene. Depletion of PBP from cell extracts by PSE-oligonucleotides abolished tRNA(Sec) transcription, which could be reconstituted by readdition of partially purified PBP. Addition of increasing amounts of recombinant human TBP to an S100 extract stimulated transcription of the tRNA(Sec), the mouse U6 snRNA and the human Y3 genes, an effect which was not observed in the case of a TATA-less tRNA gene. Purified human TFIIA strongly stimulated tRNA(Sec) transcription in a fashion depending on the concentration of TBP. Surprisingly, partially purified TFIIIC was shown to be dispensable for transcription in vitro and unable to bind the B-block of this gene in vitro, although its sequence matches the consensus for this element. Collectively, these data suggest that the mechanism by which transcription complexes are formed on the tRNA(Sec) gene is dramatically different from that observed for classical tRNA genes and much more resembles that observed for externally controlled pol III genes.
1993
Myslinski E, Schuster C, Huet J, Sentenac A, Krol A, Carbon P
Point mutations 5' to the tRNA selenocysteine TATA box alter RNA polymerase III transcription by affecting the binding of TBP Article de journal
Dans: Nucleic Acids Res, vol. 21, no. 25, p. 5852-5858, 1993, ISBN: 8290344, (0305-1048 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Amino Acid-Specific/*genetics/metabolism Recombinant Proteins/metabolism Support, Animals Base Sequence DNA/metabolism DNA-Binding Proteins/*metabolism Molecular Sequence Data *Point Mutation Protein Binding RNA Polymerase III/*metabolism RNA, Genetic Xenopus laevis, Non-U.S. Gov't *TATA Box TATA-Box Binding Protein Transcription Factors/*metabolism *Transcription, Transfer, Unité ARN
@article{,
title = {Point mutations 5' to the tRNA selenocysteine TATA box alter RNA polymerase III transcription by affecting the binding of TBP},
author = {E Myslinski and C Schuster and J Huet and A Sentenac and A Krol and P Carbon},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8290344},
isbn = {8290344},
year = {1993},
date = {1993-01-01},
journal = {Nucleic Acids Res},
volume = {21},
number = {25},
pages = {5852-5858},
abstract = {The selenocysteine tRNA(Sec) gene possesses two external promoter elements, one of which is constituted by a strong TATA box. Point mutant analysis performed in this study led to the conclusion that the functional TATA promoter actually encompasses the sequence -34 GGGTATAAAAGG-23. Individual changes at T-31 do not affect transcription much. Position T-29 is less permissive to mutation since transversion to a G, for example, is less well tolerated than at T-31. Interestingly, a double point mutation, converting GG(-33/-32) to TT, causes abrogation of transcription in vivo and severe reduction of transcription in vitro with human TBP. Therefore, data obtained underscore the fact that, in the Xenopus tRNA(Sec), these two Gs are an integral part of the TATA promoter. Gel retardation experiments indicate that the GG to TT substitution, which led human TBP to lose its ability to support efficient transcription in vitro, correlates with the appearance of an altered pattern of retarded complexes. Altogether, the data presented in this report support a model in which TBP interacts directly with the TATA element of the tRNA(Sec) gene, in contrast to the type of interaction proposed for classical TATA-less tRNA genes.},
note = {0305-1048
Journal Article},
keywords = {Amino Acid-Specific/*genetics/metabolism Recombinant Proteins/metabolism Support, Animals Base Sequence DNA/metabolism DNA-Binding Proteins/*metabolism Molecular Sequence Data *Point Mutation Protein Binding RNA Polymerase III/*metabolism RNA, Genetic Xenopus laevis, Non-U.S. Gov't *TATA Box TATA-Box Binding Protein Transcription Factors/*metabolism *Transcription, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The selenocysteine tRNA(Sec) gene possesses two external promoter elements, one of which is constituted by a strong TATA box. Point mutant analysis performed in this study led to the conclusion that the functional TATA promoter actually encompasses the sequence -34 GGGTATAAAAGG-23. Individual changes at T-31 do not affect transcription much. Position T-29 is less permissive to mutation since transversion to a G, for example, is less well tolerated than at T-31. Interestingly, a double point mutation, converting GG(-33/-32) to TT, causes abrogation of transcription in vivo and severe reduction of transcription in vitro with human TBP. Therefore, data obtained underscore the fact that, in the Xenopus tRNA(Sec), these two Gs are an integral part of the TATA promoter. Gel retardation experiments indicate that the GG to TT substitution, which led human TBP to lose its ability to support efficient transcription in vitro, correlates with the appearance of an altered pattern of retarded complexes. Altogether, the data presented in this report support a model in which TBP interacts directly with the TATA element of the tRNA(Sec) gene, in contrast to the type of interaction proposed for classical TATA-less tRNA genes.
1992
Murgo S, Krol A, Carbon P
The differential transcriptional activity of two amphibian U1 small-nuclear RNA genes correlates with structural differences in the proximal sequence element Article de journal
Dans: Eur J Biochem, vol. 203, no. 3, p. 443-447, 1992, ISBN: 1735429, (0014-2956 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Ambystoma Animals Base Sequence Microinjections Molecular Sequence Data Mutagenesis, Genetic Xenopus laevis, Non-U.S. Gov't *Transcription, Nucleic Acid Sequence Homology, Nucleic Acid Support, Site-Directed Phylogeny RNA, Small Nuclear/*genetics *Regulatory Sequences, Unité ARN
@article{,
title = {The differential transcriptional activity of two amphibian U1 small-nuclear RNA genes correlates with structural differences in the proximal sequence element},
author = {S Murgo and A Krol and P Carbon},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1735429},
isbn = {1735429},
year = {1992},
date = {1992-01-01},
journal = {Eur J Biochem},
volume = {203},
number = {3},
pages = {443-447},
abstract = {We previously analyzed the transcription of an axolotl U1 small-nuclear RNA (snRNA) gene (AmU1) by microinjection into Xenopus laevis oocytes. In such an assay, AmU1 showed a low template activity compared to that of an X. laevis U1 snRNA gene (XlU1B2). Swapping the proximal sequence element (PSE) with that of XlU1B2 was required for AmU1 to acquire a transcription level equal to that of XlU1B2. In the present work, we examine the functional importance of the nucleotides that are common or different in both PSEs with the aim of identifying which nucleotides within the Xenopus U1 PSE are critical for this enhancement of Ambystoma mexicanum U1 snRNA transcription. The PSE mutation analysis showed that the central, phylogenetically conserved C-58/C-57 doublet is absolutely required for U1 promoter activity. In the 3' portion of this element, a CGC to ATG change (positions -54/-52) which partially restores the XlU1B2 PSE sequence, enables the AmU1 gene to gain the same transcriptional activity as XlU1B2. Remarkably, in this clustered point mutation, the sole C-54 to A-54 change is sufficient to obtain this increased level. Therefore, the activity of the AmU1 gene in injected Xenopus oocytes is strongly affected by a single sequence difference between AmU1 and XlU1B2 PSEs. This finding underscores the crucial importance of the nucleotide identity at position -54 to the function of the Xenopus U1 PSE.},
note = {0014-2956
Journal Article},
keywords = {Ambystoma Animals Base Sequence Microinjections Molecular Sequence Data Mutagenesis, Genetic Xenopus laevis, Non-U.S. Gov't *Transcription, Nucleic Acid Sequence Homology, Nucleic Acid Support, Site-Directed Phylogeny RNA, Small Nuclear/*genetics *Regulatory Sequences, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
We previously analyzed the transcription of an axolotl U1 small-nuclear RNA (snRNA) gene (AmU1) by microinjection into Xenopus laevis oocytes. In such an assay, AmU1 showed a low template activity compared to that of an X. laevis U1 snRNA gene (XlU1B2). Swapping the proximal sequence element (PSE) with that of XlU1B2 was required for AmU1 to acquire a transcription level equal to that of XlU1B2. In the present work, we examine the functional importance of the nucleotides that are common or different in both PSEs with the aim of identifying which nucleotides within the Xenopus U1 PSE are critical for this enhancement of Ambystoma mexicanum U1 snRNA transcription. The PSE mutation analysis showed that the central, phylogenetically conserved C-58/C-57 doublet is absolutely required for U1 promoter activity. In the 3' portion of this element, a CGC to ATG change (positions -54/-52) which partially restores the XlU1B2 PSE sequence, enables the AmU1 gene to gain the same transcriptional activity as XlU1B2. Remarkably, in this clustered point mutation, the sole C-54 to A-54 change is sufficient to obtain this increased level. Therefore, the activity of the AmU1 gene in injected Xenopus oocytes is strongly affected by a single sequence difference between AmU1 and XlU1B2 PSEs. This finding underscores the crucial importance of the nucleotide identity at position -54 to the function of the Xenopus U1 PSE.
