Affinity purification of transcription factor IIA from HeLa cell nuclear extracts.

Usuda, Yoshihiro; Kubota, Akatsuki; Berk, Andarnold J.; Handa, Hiroshi

doi:10.1002/j.1460-2075.1991.tb07767.x

Cited by 42 publications

(38 citation statements)

References 41 publications

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“…Earlier studies (Usuda et al 1991;Cortes et al 1992;Coulombe et al 1992) demonstrated that TFIIA interacts with the TBP. Despite the fact that an interaction between TBP and TFIIA could be demonstrated, we have consistently observed that transcription reactions reconstituted with TBP are unaffected by TFIIA (Fig.…”

Section: Tfiia and Transcriptionmentioning

confidence: 93%

Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIID-mediated transcription.

Sun¹,

Ma²,

Sheldon³

et al. 1994

Genes Dev.

109

110

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Section: Tfiia and Transcriptionmentioning

confidence: 93%

Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIID-mediated transcription.

Sun¹,

Ma²,

Sheldon³

et al. 1994

Genes Dev.

109

110

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“…Since no other polypeptide that contained the transcriptionstimulatory activity could be renatured and the polypeptides required to reconstitute the stimulatory effect on TBP DNA binding activity coeluted with the transcription-stimulatory activity (Fig. 1A) 13-kDa polypeptides (39) and from wheat and HeLa cells as a 35-kDa polypeptide (6,46). TFIIA has also been identified in S. cerevisiae, but its polypeptide composition is still unknown (4,18).…”

Section: Methodsmentioning

confidence: 99%

Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ.

Cortés¹,

Flores²,

Reinberg³

1992

Mol. Cell. Biol.

125

154

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The previously described transcription factor IIA (TFIIA) protein fraction was separated into two factors that affect transcription, TFIIA and TFIIJ. TFIIA was found to have a stimulatory effect, and TFIIJ was found to be required for transcription. The tein fraction is composed of at least two separate components, TFIIA and TFIIJ. We found that TFIIA acted to increase transcription, whereas transcription was dependent on TFIIJ. The TFIIA-mediated stimulation of transcription was dependent on the source of TFIID. TFIIA was found to have a stimulatory effect only when the transcription assays contained endogenous HeLa cell TFIID. Transcription assays reconstituted with recombinant yeast TBP (yTBP) or human TBP (hTBP) were independent of TFIIA. MATERIALS AND METHODSSpecific transcription assay and transcription factors. Transcription reactions and product analysis were performed as previously described (36). The DNA template used was ApML(C2AT), which contains the adenovirus major late promoter (Ad-MLP) sequences extending from -50 to +11 For assays measuring TFIIA activity, the TFIID protein fraction was used and the TFIIA protein fraction was omitted and replaced by column fractions. For assays measuring TFIIJ activity, h-or yTBP was used in lieu of TFIID and the TFIIE/F/H protein fraction was replaced by more purified factors (14a). The TFIIJ protein fraction used was derived from either the phosphocellulose 0.1 M KCI or TFIIG (45) protein fractions, as indicated in the figure legends. TFIIJ derived from the 0.1 M KCI fraction was purified as described below. TFIIJ derived from the TFIIG

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“…A, p160 interacts with the largest subunit of pol II. pol II was purified from HeLa cell nuclear pellet as described (37). pol II, GST-p14, and GST (200 ng each) were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membrane, and renatured as described (38).…”

Section: Fig 4 Mapping the Region Of P160 Important For Dsif Activimentioning

confidence: 99%

Structure and Function of the Human Transcription Elongation Factor DSIF

Yamaguchi¹,

Wada

Watanabe

et al. 1999

Journal of Biological Chemistry

127

155

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5,6-Dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB)is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). We have previously identified and purified a novel transcription elongation factor, termed DSIF (for DRB sensitivity-inducing factor), that makes transcription sensitive to DRB. DSIF is composed of 160-and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4. DSIF may either repress or stimulate transcription in vitro, depending on conditions, but its physiological function remains elusive. Here we characterize the structure and function of DSIF p160. p160 is shown to be a ubiquitous nuclear protein that forms a stable complex with p14 and interacts directly with the pol II largest subunit. Mutation analysis of p160 is used to identify structural features essential for its in vitro activity and to map the domains required for its interaction with p14 and pol II. Finally, a p160 mutant that represses DSIF activity in a dominant-negative manner is identified and used to demonstrate that DSIF represses transcription from various promoters in vivo.The nucleoside analog DRB 1 is a classic inhibitor of transcription elongation by pol II (reviewed in Ref. 1). Although it has been used for more than three decades, its mode of action has long been a mystery. DRB is unique in that it shows no effect on transcription reconstituted with purified general transcription factors (GTFs) and pol II, whereas it potently represses transcription in cruder systems or in vivo (2-4). Therefore, one or more factors apart from GTFs and pol II appear to be involved in DRB-sensitive transcription (1). Such putative factors must play general roles in pol II transcription in vivo because DRB affects most of the class II genes (4 -6).Recently, we and others have identified two elongation factors essential for DRB-sensitive transcription. One of them, positive transcription elongation factor b (P-TEFb), is a protein kinase that phosphorylates the pol II C-terminal domain (CTD) in a DRB-sensitive fashion (7-12). The CTD phosphorylation likely plays a pivotal role in pol II elongation and may be relevant to the P-TEFb function. The other, DSIF, has been purified from HeLa cell nuclear extract, based on its ability to induce DRB-sensitivity in vitro (3). DSIF is composed of 160-and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4 (3, 13-15). DSIF/Spt4-Spt5 genetically and physically interacts with pol II, and thus may directly regulate pol II processivity (3, 16).The function of DSIF in the absence of DRB remains obscure. Small amounts of DSIF, when added back to a DSIF-depleted transcription system, repress transcription only in the presence of DRB, without affecting transcription in its absence (3). Higher doses of DSIF, however, repress transcription even in the absence of DRB (3). In contrast, under limiting concentrations of NTPs, DSIF stimulates transcription elongation in the absence of DRB (3). Genetic a...

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Affinity purification of transcription factor IIA from HeLa cell nuclear extracts.

Cited by 42 publications

References 41 publications

Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIID-mediated transcription.

Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIID-mediated transcription.

Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ.

Structure and Function of the Human Transcription Elongation Factor DSIF

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