Basal and regulated transcription in Archaea

2001 ◽  
Vol 29 (4) ◽  
pp. 392-395 ◽  
Author(s):  
S. D. Bell ◽  
C. P. Magill ◽  
S. P. Jackson
Keyword(s):  
In Vitro Transcription ◽  
Tata Box ◽  
Basal Transcription ◽  
General Transcription Factors ◽  
General Transcription Factor ◽  
Promoter Recognition ◽  
Transcription In Vitro ◽  
Rnap Ii ◽  
Tata Box Binding Protein

The basal transcription machinery of Archaea is fundamentally related to the eucaryal RNA polymerase (RNAP) II apparatus. In addition to a 12-subunit RNAP, Archaea possess two general transcription factors, the activities of which are required for accurate and efficient in vitro transcription. These factors, TBP and TFB, are homologues of the eucaryal TATA-box binding protein and TFIIB respectively. Archaea also possess TFE, a homologue of the eucaryal RNAP II general transcription factor TFIIE. Although not absolutely required for transcription in vitro, TFE nonetheless plays a stimulatory role under conditions where promoter recognition by TBP is sub-optimal. The basal transcription apparatus of Archaea is closely related to that of Eucarya but archaeal transcriptional regulators resemble those of bacteria. The mode of action of two such regulators has been characterized to determine how these ‘bacterial-like’ regulators impinge on the ‘eucaryal-like’ basal machinery.

scholarly journals Identification of a Conserved Archaeal RNA Polymerase Subunit Contacted by the Basal Transcription Factor TFB

2001 ◽  
Vol 276 (50) ◽  
pp. 46693-46696 ◽  
Author(s):  
Christine P. Magill ◽  
Stephen P. Jackson ◽  
Stephen D. Bell
Keyword(s):  
Rna Polymerase ◽  
General Transcription Factors ◽  
Bacterial Rna ◽  
Evolutionarily Conserved ◽  
Archaeal Transcription ◽  
Rnap Ii ◽  
Biochemical Analyses ◽  
Tata Box Binding Protein ◽  
Two Hybrid

Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promotersin vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (ω-subunit).

The in vitro transcription of a rainbow trout (Salmo gairdnerii) protamine gene. II. Controlled mutation of the cap site region

1985 ◽  
Vol 5 (2) ◽  
pp. 113-120 ◽  
Author(s):  
Jacek M. Jankowski ◽  
Gordon H. Dixon
Keyword(s):  
In Vitro Transcription ◽  
Tata Box ◽  
Fusion Genes ◽  
Complementary Strand ◽  
Coding Region ◽  
Cryptic Promoter ◽  
Cell Lysate ◽  
Multiple Transcripts ◽  
Transcription In Vitro

A series of pJasmids containing new fusion genes in which the trout protamine gene is placed under the control of the complete herpes virus (HSV-I) tk promoter Pvu II-Bgl II fragment (pMg), or a shortened thymidine kinase (tk) promoter in which the region between the TATA box and the cap site is altered by using the Pvu II-Mlu I fragment (pM7), have been constructed. An additional recombinant plasmid was constructed in which the Bgl II-Ava II fragment of the protamine gene containing the entire protamine promoter but missing the protamine coding region was cloned into pBR322 between the Xho II 1666 and Hind III sites (pP5). For in vitro transcription, a HeLa cell lysate system was prepared and the RNA transcription products, after glyoxalation, were electrophoretically analyzed on 5% polyacrylamide gels. In constructing pM8 the DNA sequence between the tk promoter and the cap site was present while in pM7 it was deleted. Similar multiple transcripts were seen in both cases, indicating that the region between the promoter and the cap site has no effect upon transcription in vitro. The multiple transcripts appear to be due to the presence of a cryptic promoter in the complementary strand of the protamine gene. The activity of this cryptic promoter has been confirmed by comparison of the transcription of plasmid pPS, in which the protamine mRNA coding region has been deleted, with a previously described plasmid, p3BRP (Jankowski 3M and Dixon GH (1984) Can. J. Biochem. Cell. Biol. 62, 291–300), containing the intact protamine gene.

scholarly journals Transcriptional activators differ in their responses to overexpression of TATA-box-binding protein.

1995 ◽  
Vol 15 (3) ◽  
pp. 1554-1563 ◽  
Author(s):  
Y Sadovsky ◽  
P Webb ◽  
G Lopez ◽  
J D Baxter ◽  
P M Fitzpatrick ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Binding Protein ◽  
Tata Box ◽  
Transcriptional Activators ◽  
Basal Transcription ◽  
Response Element ◽  
Synergistic Activation ◽  
Endogenous Genes ◽  
Tata Box Binding Protein

We investigated how overexpression of human TATA-box-binding protein (TBP) affects the action of estrogen receptor (ER) and compared the response with that of other activators. When ER activates a simple promoter, consisting of a response element and either the collagenase or tk TATA box, TBP overexpression potentiates transcription. TBP potentiates only estrogen-induced and not basal transcription and does so independent of spacing between response element and TATA box. TBP overexpression also reduces autoinhibition by overexpressed ER, suggesting that one target of the autoinhibition may be TBP itself. Both AF-1 and AF-2 domains of ER are potentiated by TBP, and each domain binds TBP in vitro. Like ER, chimeric GAL4/VP16 and GAL4/Tat activators are also potentiated by TBP, as is the synergistic activation by ER and GAL4/VP16 on a complex promoter. Unlike ER, GAL4/Sp1 and GAL4/NF-I become less potent when TBP is overexpressed. Furthermore, synergy between ER and Sp1 or between ER and NF-I, whether these are supplied by transfected GAL4 fusions or by the endogenous genes, is inhibited by TBP overexpression. Thus, ER resembles VP16 in response to TBP overexpression and is different from Sp1 and NF-I, which predominate over ER in setting the response on complex promoters.

scholarly journals MOT1 Can Activate Basal Transcription In Vitro by Regulating the Distribution of TATA Binding Protein between Promoter and Nonpromoter Sites

1999 ◽  
Vol 19 (4) ◽  
pp. 2835-2845 ◽  
Author(s):  
Tamara A. Muldrow ◽  
Allyson M. Campbell ◽  
P. Anthony Weil ◽  
David T. Auble
Keyword(s):  
Transcriptional Control ◽  
Binding Protein ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Wild Type ◽  
Basal Transcription ◽  
Nuclear Extracts ◽  
Transcription In Vitro

ABSTRACT MOT1 is an ATPase which can dissociate TATA binding protein (TBP)-DNA complexes in a reaction requiring ATP hydrolysis. Consistent with this observation, MOT1 can repress basal transcription in vitro. Paradoxically, however, some genes, such as HIS4, appear to require MOT1 as an activator of transcription in vivo. To further investigate the function of MOT1 in basal transcription, we performed in vitro transcription reactions using yeast nuclear extracts depleted of MOT1. Quantitation of MOT1 revealed that it is an abundant protein, with nuclear extracts from wild-type cells containing a molar excess of MOT1 over TBP. Surprisingly, MOT1 can weakly activate basal transcription in vitro. This activation by MOT1 is detectable with amounts of MOT1 that are approximately stoichiometric to TBP. With amounts of MOT1 similar to those present in wild-type nuclear extracts, MOT1 behaves as a weak repressor of basal transcription. These results suggest that MOT1 might activate transcription via an indirect mechanism in which limiting TBP can be liberated from nonpromoter sites for use at promoters. In support of this idea, excess nonpromoter DNA sequesters TBP and represses transcription, but this effect can be reversed by addition of MOT1. These results help to reconcile previous in vitro and in vivo results and expand the repertoire of transcriptional control strategies to include factor-assisted redistribution of TBP between promoter and nonpromoter sites.

The general transcription factor RAP30 binds to RNA polymerase II and prevents it from binding nonspecifically to DNA

1992 ◽  
Vol 12 (1) ◽  
pp. 30-37
Author(s):  
M T Killeen ◽  
J F Greenblatt
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Small Subunit ◽  
Glutathione S Transferase ◽  
General Transcription Factor ◽  
Indirect Consequence ◽  
Transcription In Vitro ◽  
Sigma 70

RAP30/74 is a human general transcription factor that binds to RNA polymerase II and is required for initiation of transcription in vitro regardless of whether the promoter has a recognizable TATA box (Z. F. Burton, M. Killeen, M. Sopta, L. G. Ortolan, and J. F. Greenblatt, Mol. Cell. Biol. 8:1602-1613, 1988). Part of the amino acid sequence of RAP30, the small subunit of RAP30/74, has limited homology with part of Escherichia coli sigma 70 (M. Sopta, Z. F. Burton, and J. Greenblatt, Nature (London) 341:410-414, 1989). To determine which sigmalike activities of RAP30/74 could be attributed to RAP30, we purified human RAP30 and a RAP30-glutathione-S-transferase fusion protein that had been produced in E. coli. Bacterially produced RAP30 bound to RNA polymerase II in the absence of RAP74. Both partially purified natural RAP30/74 and recombinant RAP30 prevented RNA polymerase II from binding nonspecifically to DNA. In addition, nonspecific transcription by RNA polymerase II was greatly inhibited by RAP30-glutathione-S-transferase. DNA-bound RNA polymerase II could be removed from DNA by partially purified RAP30/74 but not by bacterially expressed RAP30. Thus, the ability of RAP30/74 to recruit RNA polymerase II to a promoter-bound preinitiation complex may be an indirect consequence of its ability to suppress nonspecific binding of RNA polymerase II to DNA.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals Entamoeba histolytica TATA-box binding protein binds to different TATA variants in vitro

FEBS Journal ◽  
2005 ◽  
Vol 272 (6) ◽  
pp. 1354-1366 ◽  
Author(s):  
Guadalupe De Dios-Bravo ◽  
Juan Pedro Luna-Arias ◽  
Ana María Riverón ◽  
José J Olivares-Trejo ◽  
César López-Camarillo ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Binding Protein ◽  
Tata Box ◽  
Tata Box Binding Protein
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