Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes

Jong Bok Yoon, Shona Murphy, Lin Bai, Zhengxin Wang, Robert G. Roeder

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Abstract

The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP- TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific factors for PSE- and B-box- containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the form used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

Original languageEnglish
Pages (from-to)2019-2027
Number of pages9
JournalMolecular and Cellular Biology
Volume15
Issue number4
DOIs
Publication statusPublished - 1995 Apr

Fingerprint

RNA Polymerase III
Small Nuclear RNA
RNA Polymerase II
Transcription Factors
TATA-Box Binding Protein
Genes
TATA-Binding Protein Associated Factors
Transcription Factor TFIIIB
pol Genes
Peptides
Cell Extracts
HeLa Cells
Recombinant Proteins
Protein Binding
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
Hot Temperature
transcription factor TFIIIC
RNA

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

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title = "Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes",
abstract = "The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP- TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific factors for PSE- and B-box- containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the form used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.",
author = "Yoon, {Jong Bok} and Shona Murphy and Lin Bai and Zhengxin Wang and Roeder, {Robert G.}",
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AU - Yoon, Jong Bok

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AU - Bai, Lin

AU - Wang, Zhengxin

AU - Roeder, Robert G.

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N2 - The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP- TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific factors for PSE- and B-box- containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the form used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

AB - The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP- TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific factors for PSE- and B-box- containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the form used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

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