TATA box-dependent protein-DNA interactions are detected on heat shock and histone gene promoters in nuclear extracts derived from Drosophila melanogaster embryos

1988 ◽  
Vol 8 (8) ◽  
pp. 3204-3214
Author(s):  
D S Gilmour ◽  
T J Dietz ◽  
S C Elgin
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Transcriptional Activation ◽  
Tata Box ◽  
Gene Promoters ◽  
Dna Interactions ◽  
Nuclear Extracts ◽  
Protein Dna Interactions ◽  
Exonuclease Digestion

We monitored protein-DNA interactions that occur on the hsp26, hsp70, histone H3, and histone H4 promoters in nuclear extracts derived from frozen Drosophila melanogaster embryos. All four of these promoters were found to be transcribed in vitro at comparable levels by extracts from both heat-shocked and non-heat-shocked embryos. Factors were detected in both types of extracts that block exonuclease digestion from a downstream site at ca. +35 and -20 base pairs from the start of transcription of all four of these promoters. In addition, factors in extracts from heat-shocked embryos blocked exonuclease digestion at sites flanking the heat shock consensus sequences of hsp26 and hsp70. Competition experiments indicated that common factors cause the +35 and -20 barriers on all four promoters in both extracts. The formation of the barriers at +35 and -20 required a TATA box but did not appear to require specific sequences downstream of +7. We suggest that the factors responsible for the +35 and -20 barriers are components whose association with the promoter precedes transcriptional activation.

scholarly journals TATA box-dependent protein-DNA interactions are detected on heat shock and histone gene promoters in nuclear extracts derived from Drosophila melanogaster embryos.

1988 ◽  
Vol 8 (8) ◽  
pp. 3204-3214 ◽  
Author(s):  
D S Gilmour ◽  
T J Dietz ◽  
S C Elgin
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Transcriptional Activation ◽  
Tata Box ◽  
Gene Promoters ◽  
Dna Interactions ◽  
Nuclear Extracts ◽  
Protein Dna Interactions ◽  
Exonuclease Digestion

We monitored protein-DNA interactions that occur on the hsp26, hsp70, histone H3, and histone H4 promoters in nuclear extracts derived from frozen Drosophila melanogaster embryos. All four of these promoters were found to be transcribed in vitro at comparable levels by extracts from both heat-shocked and non-heat-shocked embryos. Factors were detected in both types of extracts that block exonuclease digestion from a downstream site at ca. +35 and -20 base pairs from the start of transcription of all four of these promoters. In addition, factors in extracts from heat-shocked embryos blocked exonuclease digestion at sites flanking the heat shock consensus sequences of hsp26 and hsp70. Competition experiments indicated that common factors cause the +35 and -20 barriers on all four promoters in both extracts. The formation of the barriers at +35 and -20 required a TATA box but did not appear to require specific sequences downstream of +7. We suggest that the factors responsible for the +35 and -20 barriers are components whose association with the promoter precedes transcriptional activation.

scholarly journals Scanning Mutagenesis of Mcm1: Residues Required for DNA Binding, DNA Bending, and Transcriptional Activation by a MADS-Box Protein

2000 ◽  
Vol 20 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Thomas B. Acton ◽  
Janet Mead ◽  
Andrew M. Steiner ◽  
Andrew K. Vershon
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Essential Gene ◽  
Dna Bending ◽  
Mads Box ◽  
Dna Interactions ◽  
Protein Dna Interactions

ABSTRACT MCM1 is an essential gene in the yeastSaccharomyces cerevisiae and is a member of the MADS-box family of transcriptional regulatory factors. To understand the nature of the protein-DNA interactions of this class of proteins, we have made a series of alanine substitutions in the DNA-binding domain of Mcm1 and examined the effects of these mutations in vivo and in vitro. Our results indicate which residues of Mcm1 are important for viability, transcriptional activation, and DNA binding and bending. Substitution of residues in Mcm1 which are highly conserved among the MADS-box proteins are lethal to the cell and abolish DNA binding in vitro. These positions have almost identical interactions with DNA in both the serum response factor-DNA and α2-Mcm1-DNA crystal structures, suggesting that these residues make up a conserved core of protein-DNA interactions responsible for docking MADS-box proteins to DNA. Substitution of residues which are not as well conserved among members of the MADS-box family play important roles in contributing to the specificity of DNA binding. These results suggest a general model of how MADS-box proteins recognize and bind DNA. We also provide evidence that the N-terminal extension of Mcm1 may have considerable conformational freedom, possibly to allow binding to different DNA sites. Finally, we have identified two mutants at positions which are critical for Mcm1-mediated DNA bending that have a slow-growth phenotype. This finding is consistent with our earlier results, indicating that DNA bending may have a role in Mcm1 function in the cell.

scholarly journals Mediators of activation of fushi tarazu gene transcription by BmFTZ-F1.

1994 ◽  
Vol 14 (5) ◽  
pp. 3013-3021 ◽  
Author(s):  
F Q Li ◽  
H Ueda ◽  
S Hirose
Keyword(s):  
Transcriptional Activation ◽  
Direct Contact ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Dna Interactions ◽  
Fushi Tarazu ◽  
Eukaryotic Sequence ◽  
Protein Dna Interactions ◽  
Dna Complex

Transcriptional activation by many eukaryotic sequence-specific regulators appears to be mediated through transcription factors which do not directly bind to DNA. BmFTZ-F1 is a silkworm counterpart of FTZ-F1, a sequence-specific activator of the fushi tarazu gene in Drosophila melanogaster. We report here the isolation of 18- and 22-kDa polypeptides termed MBF1 and MBF2, respectively, that form a heterodimer and mediate activation of in vitro transcription from the fushi tarazu promoter by BmFTZ-F1. Neither MBF1, MBF2, nor a combination of them binds to DNA. MBF1 interacts with BmFTZ-F1 and stabilizes the BmFTZ-F1-DNA complex. MBF1 also makes direct contact with TATA-binding protein (TBP). Both MBF1 and MBF2 are necessary to form a complex between BmFTZ-F1 and TBP. We propose a model in which MBF1 and MBF2 form a bridge between BmFTZ-F1 and TBP and mediate transactivation by stabilizing the protein-DNA interactions.

scholarly journals HSP90 Interacts with and Regulates the Activity of Heat Shock Factor 1 in Xenopus Oocytes

1998 ◽  
Vol 18 (9) ◽  
pp. 4949-4960 ◽  
Author(s):  
Adnan Ali ◽  
Steven Bharadwaj ◽  
Ruth O’Carroll ◽  
Nick Ovsenek
Keyword(s):  
Heat Shock ◽  
Transcriptional Activation ◽  
Cellular Signaling ◽  
Heat Shock Factor ◽  
Heat Shock Factor 1 ◽  
Heat Shock Element ◽  
Nuclear Extracts ◽  
Multistep Process

ABSTRACT Transcriptional activation of heat shock genes is a reversible and multistep process involving conversion of inactive heat shock factor 1 (HSF1) monomers into heat shock element (HSE)-binding homotrimers, hyperphosphorylation, and further modifications that induce full transcriptional competence. HSF1 is controlled by multiple regulatory mechanisms, including suppression by additional cellular factors, physical interactions with HSP70, and integration into different cellular signaling cascades. However, the signaling mechanisms by which cells respond to stress and control the HSF1 activation-deactivation pathway are not known. Here we demonstrate that HSP90, a cellular chaperone known to regulate several signal transduction molecules and transcription factors, functions in the regulation of HSF1. The existence of HSF1-HSP90 heterocomplexes was shown by coimmunoprecipitation of HSP90 with HSF1 from unshocked and heat-shocked nuclear extracts, recognition of HSF1-HSE complexes in vitro by using HSP90 antibodies (Abs), and recognition of HSF1 in vivo by HSP90 Abs microinjected directly into oocyte nuclei. The functional impact of HSP90-HSF1 interactions was analyzed by using two strategies: direct nuclear injection of HSP90 Abs and treatment of cells with geldanamycin (GA), an agent that specifically blocks the chaperoning activity of HSP90. Both HSP90 Abs and GA delayed the disassembly of HSF1 trimers during recovery from heat shock and specifically inhibited heat-induced transcription from a chloramphenicol acetyltransferase reporter construct under control of the hsp70 promoter. HSP90 Abs activated HSE binding in the absence of heat shock, an effect that could be reversed by subsequent injection of purified HSP90. GA did not activate HSE binding under nonshock conditions but increased the quantity of HSE binding induced by heat shock. On the basis of these findings and the known properties of HSP90, we propose a new regulatory model in which HSP90 participates in modulating HSF1 at different points along the activation-deactivation pathway, influencing the interconversion between monomeric and trimeric conformations as well as transcriptional activation. We also put forth the hypothesis that HSP90 links HSF1 to cellular signaling molecules coordinating the stress response.

scholarly journals Mediators of activation of fushi tarazu gene transcription by BmFTZ-F1

1994 ◽  
Vol 14 (5) ◽  
pp. 3013-3021
Author(s):  
F Q Li ◽  
H Ueda ◽  
S Hirose
Keyword(s):  
Transcriptional Activation ◽  
Direct Contact ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Dna Interactions ◽  
Fushi Tarazu ◽  
Eukaryotic Sequence ◽  
Protein Dna Interactions ◽  
Dna Complex

Transcriptional activation by many eukaryotic sequence-specific regulators appears to be mediated through transcription factors which do not directly bind to DNA. BmFTZ-F1 is a silkworm counterpart of FTZ-F1, a sequence-specific activator of the fushi tarazu gene in Drosophila melanogaster. We report here the isolation of 18- and 22-kDa polypeptides termed MBF1 and MBF2, respectively, that form a heterodimer and mediate activation of in vitro transcription from the fushi tarazu promoter by BmFTZ-F1. Neither MBF1, MBF2, nor a combination of them binds to DNA. MBF1 interacts with BmFTZ-F1 and stabilizes the BmFTZ-F1-DNA complex. MBF1 also makes direct contact with TATA-binding protein (TBP). Both MBF1 and MBF2 are necessary to form a complex between BmFTZ-F1 and TBP. We propose a model in which MBF1 and MBF2 form a bridge between BmFTZ-F1 and TBP and mediate transactivation by stabilizing the protein-DNA interactions.

Protein–DNA interactions at theSulfolobusspindle-shaped virus-1 (SSV1) T5 and T6 gene promoters

2007 ◽  
Vol 53 (9) ◽  
pp. 1076-1083 ◽  
Author(s):  
Sohail A. Qureshi
Keyword(s):  
Dna Replication ◽  
Chemical Modification ◽  
Gene Promoters ◽  
Dna Interactions ◽  
Viral Dna ◽  
Interacting Protein ◽  
Viral Dna Replication ◽  
Protein Dna Interactions ◽  
Sulfolobus Shibatae

Ultraviolet irradiation upregulates transcription from the Sulfolobus spindle-shaped virus 1 (SSV-1) T5, Tind, and T6 genes promoters and also triggers viral DNA replication, but nothing is known about the proteins involved in this process. A notable feature of T5 and T6 promoters is that they contain 4 copies of a highly conserved DNA sequence 5′-ATAGATAGAGT-3′; 2 copies of this repeat are found in tandem upstream of the A-box, whereas 2 additional tandem copies span the initiator region from which transcription originates. By employing electrophoretic mobility gel-shift assays (EMSAs) and chemical modification interference analyses, I have identified a protein STRIP (SSV-1 T5/T6 region-interacting protein) in Sulfolobus shibatae extract that binds specifically to this sequence. Unique to S. shibatae, STRIP induces a 28° bend in DNA. Surprisingly, despite the fact that STRIP binding masks the initiator region and can potentially interfere with preinitiation complex assembly, it does not appear to effect transcription driven from T5 and T6 promoters in vitro. Based on these results, I discuss the potential roles of STRIP in T5 and T6 transcription and in initiating SSV-1 DNA replication.

Modular recognition of 5-base-pair DNA sequence motifs by human heat shock transcription factor

1991 ◽  
Vol 11 (7) ◽  
pp. 3504-3514
Author(s):  
N F Cunniff ◽  
J Wagner ◽  
W D Morgan
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Gene Promoter ◽  
Heat Shock Transcription Factor ◽  
Heat Shock Gene ◽  
Gene Promoters ◽  
Sequence Motifs ◽  
Binding Modes ◽  
Heat Shock Element

We investigated the recognition of the conserved 5-bp repeated motif NGAAN, which occurs in heat shock gene promoters of Drosophila melanogaster and other eukaryotic organisms, by human heat shock transcription factor (HSF). Extended heat shock element mutants of the human HSP70 gene promoter, containing additional NGAAN blocks flanking the original element, showed significantly higher affinity than the wild-type promoter element for human HSF in vitro. Protein-DNA contact positions were identified by hydroxyl radical protection, diethyl pyrocarbonate interference, and DNase I footprinting. New contacts in the mutant HSE constructs corresponded to the locations of additional NGAAN motifs. The pattern of binding indicated the occurrence of multiple DNA binding modes for HSF with the various constructs and was consistent with an oligomeric, possibly trimeric, structure of the protein. In contrast to the improved binding, the extended heat shock element mutant constructs did not exhibit dramatically increased heat-inducible transcription in transient expression assays with HeLa cells.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

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.

Two transcriptional activators, CCAAT-box-binding transcription factor and heat shock transcription factor, interact with a human hsp70 gene promoter

1987 ◽  
Vol 7 (3) ◽  
pp. 1129-1138
Author(s):  
W D Morgan ◽  
G T Williams ◽  
R I Morimoto ◽  
J Greene ◽  
R E Kingston ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Gene Promoter ◽  
Heat Shock Transcription Factor ◽  
Transcriptional Activators ◽  
Hsp70 Gene ◽  
Nuclear Extracts ◽  
Ccaat Box ◽  
Human Hsp70

We characterized the activity of a human hsp70 gene promoter by in vitro transcription. Analysis of 5' deletion and substitution mutants in HeLa nuclear extracts showed that the basal activity of the promoter depends primarily on a CCAAT-box sequence located at -65. A protein factor, CCAAT-box-binding transcription factor (CTF), was isolated from HeLa nuclear extracts and shown to be responsible for stimulation of transcription in a reconstituted in vitro system. DNase I footprinting revealed that CTF interacts with two CCAAT-box elements located at -65 and -147 of the human hsp70 promoter. An additional binding activity, heat shock transcription factor (HSTF), which interacted with the heat shock element, was also identified in HeLa extract fractions. This demonstrates that the promoter of this human hsp70 gene interacts with at least two positive transcriptional activators, CTF, which is required for CCAAT-box-dependent transcription as in other promoters such as those of globin and herpes simplex virus thymidine kinase genes, and HSTF, which is involved in heat inducibility.

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