Promoter Activation via a Cyclic AMP Response Element in Vitro

Wolner, Branden S.; Gralla, Jay D.

doi:10.1074/jbc.272.51.32301

Cited by 10 publications

(24 citation statements)

References 41 publications

(74 reference statements)

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“…This is suggested most prominently by comparing properties of the two prototype adenovirus promoters, the major late (ML) and the E4. Both in vivo and in vitro studies have shown that ML is the stronger basal promoter while E4 is more responsive to activators (7,8,10,12,13,14,(15)(16)(17)37). One possibility is that this is a simple consequence of activators compensating for the low basal transcription specified by the "weaker" E4 TATA and Inr sequences.…”

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confidence: 99%

Roles for Non-TATA Core Promoter Sequences in Transcription and Factor Binding

Wolner

Gralla

2000

Molecular and Cellular Biology

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Sequence blocks within the core region were swapped among RNA polymerase II promoters to explore effects on transcription in vitro. The pair of blocks flanking TATA strongly influenced general transcription, with an additional effect on promoter activation. These flanking elements induced a change in the ratio of activated to basal transcription, whereas swapping TATA and initiator sequences only altered general transcription levels. Swapping the flanking blocks influenced binding by general transcription factors TBP and TFIIB. The results suggest that the architecture of the extended core sequence is important in determining promoter-specific effects on both general transcription levels and the tightness of regulation.Accurate initiation of transcription by RNA polymerase II (pol II) requires the formation of a large, multiprotein complex at the gene promoter (reviewed in references 11, 26, and 29). A typical core promoter is approximately 60 bp long and extends to just downstream of the transcription start site. Recognition elements include the TATA box element located 25 to 30 bp upstream of the start site and an initiator (Inr) element spanning the start site (reviewed in references 11 and 33). These two elements, either singly or in combination, are thought to be sufficient to drive basal levels of transcription (11,19,33,34). Sometimes a downstream promoter element exists just beyond the core (2). Virtually all cellular promoters are thought to be associated with binding sites for activator proteins. These activators can be constitutive or inducible and typically bind upstream to help recruit the transcription machinery to the core promoter elements (27,35). The core promoter region contains the interaction sites for several of the general transcription factors (11,33). A weak, sequence-specific TFIIB binding site exists immediately upstream of the TATA box element (21). Other factors collectively contact nearly the entire extent of the core promoter sequences (5,20,22,23,28). With the possible exception of the upstream TFIIB site, which may influence basal transcription levels, these non-TATA and non-Inr sequences have not been implicated in control of transcription. The sequences between the TATA box and transcription start site are thought only to provide appropriate spacing (11).Although activation determinants have recently been found within Inr (3), the specificity of activation has been largely associated with distal regions. That is, removal of activation sites leads to core promoters that function in basal transcription with RNA levels depending on the "strength" of TATA and/or Inr (11,27,35).Nonetheless, prior studies are consistent with a role of the core sequences in setting the quantitative response to activators. This is suggested most prominently by comparing properties of the two prototype adenovirus promoters, the major late (ML) and the E4. Both in vivo and in vitro studies have shown that ML is the stronger basal promoter while E4 is more responsive to activators (7,8,10,12,13,14,(15)(1...

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confidence: 99%

Roles for Non-TATA Core Promoter Sequences in Transcription and Factor Binding

Wolner

Gralla

2000

Molecular and Cellular Biology

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“…The template was preincubated with nuclear extract and recombinant proteins for 10 min to allow the formation of preinitiation complexes. Then, two of the four nucleotides required for transcription were added for 2 min to allow initiation of transcripts, after which 0.02% Sarkosyl was added to inhibit new complex formation, together with the remaining nucleotides to allow completion of transcripts initiated by recruited polymerase complexes (10,28). CAD-G4, CRG, or CRG-S133A were equally effective in recruiting a polymerase complex to the promoter and stimulating transcript synthesis.…”

Section: Resultsmentioning

confidence: 99%

“…The mRNA synthesized was quantitated by primer extension analysis and was corrected for the relative amount of an internal RNA standard added in the transcription stop mix. For abortive initiation (29,30), the nuclear extract was passed over Sephadex G50 to remove contaminating nucleotides (10). Abortive initiation reactions were performed in transcription buffer and included 1 mM dinucleotide substrate (ApG)͞10 M dATP͞1 M [␣- otide substrate and trinucleotide product were separated on a 23% polyacrylamide͞7 M urea gel.…”

Section: Methodsmentioning

confidence: 99%

“…We previously showed that stimulation of in vitro transcription by cAMP-activatable PKA required a CRE site in the template and could be inhibited by a PKA-specific inhibitor peptide or by the addition of phosphatase but PKA did not affect binding of CREB (12). Additional in vitro transcription studies by others suggested that factors associated with ATF͞CRE sites could promote any (7,9,10) or all (11) of the steps in transcription initiation in response to cAMP. However, interpretation of these studies is limited by the possible complication that PKA phosphorylates and changes the activity of other proteins in the nuclear extracts.…”

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“…Previous studies, based on the binding of heterogeneous activating transcription factor (ATF)͞CREB proteins to CRE sites, have produced conf licting results (7)(8)(9)(10)(11)(12). Early studies using footprinting demonstrated that the presence of an ATF͞CRE site in a promoter produced an extended footprint, caused by a complex containing RNA polymerase II and TFIIB, TFIID, and TFIIE (7,8).…”

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