Two catabolite activator protein molecules bind to the galactose promoter region of
            <i>Escherichia coli</i>
            in the presence of RNA polymerase

Shanblatt, S H; Revzin, Arnold

doi:10.1073/pnas.80.6.1594

Cited by 44 publications

(24 citation statements)

References 27 publications

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“…Lane d shows the protection due to both RNA polymerase and cAMP-CRP: clearly a tight complex is formed that gives extensive protection from + 17 to -65. Comparison of this footprint with published data for the CRP-induced binding of RNA polymerase to gal P1 [11,16,17] shows that, in this case, RNA polymerase is indeed bound at P1. To confirm this, we compared the complexes formed between cAMP-CRP, RNA polymerase and gal DNA carrying either the mutation at -12 or a mutation at -19 that inactivates P2 such that P1 is the sole functional promoter [6,8].…”

supporting

confidence: 53%

“…Lane a shows the pattern of bands formed when end-labelled Pstl-HindlII DNA fragments covering the gal promoter region were treated with DNase I. Lane b shows that cAMP-CRP alone binds normally giving a footprint between -30 and -50 identical to that reported [11,15,16]. Lane c shows that RNA polymerase alone gives a very weak footprint, unlike that found when RNA polymerase binds to P2 [11,16,17], confirming that the complex between RNA polymerase and P3 is unstable.…”

mentioning

confidence: 87%

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Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein

1987

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We report in vitro studies of the interactions between purified E. coli RNA polymerase and DNA from the regulatory region of the E. coli galactose operon which carries a point mutation that simultaneously stops transcription initiation at the two normal start points, $1 and $2. In the presence of this point mutation, transcription initiates at a third start point 14/15 bp downstream of $1, showing that inactivation of the two normally active promoters, P1 and P2, unmasks a third weaker promoter, P3. Transcription initiation in the gal operon is normally regulated by the cyclic AMP receptor protein, CRP, that binds to the gal regulatory region and switches transcription from P2 to PI. With the point mutation, CRP binding switches transcription from P3 to P1, although the formation of transcriptionally competent complexes at P1 is very slow. The results are discussed with respect to the mechanism of transcription activation by the CRP factor and the similarities between the regulatory regions of the galactose and lactose operons.

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supporting

confidence: 53%

mentioning

confidence: 87%

Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein

1987

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“…It has recently been suggested that the transcription initiation complex at the gal promoter may contain an additional CRP molecule which could bind between -50 and -70 and could stabilize the active CRP-RNA polymerase complex (23). It is clear from the data presented here that no specific gal DNA sequences preceding -59 are required for CRP-dependent transcription.…”

Section: Discussionmentioning

confidence: 64%

Interactions of RNA polymerase and the cyclic AMP receptor protein on DNA of theE. coligalactose operon

Taniguchi

Crombrugghe

1983

Nucl Acids Res

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We have examined the interaction site on gal DNA for the cyclic AMP receptor protein and RNA polymerase when both are present together to form a stable initiation complex at the P gal promoter. Substitution of the bases to the left of -60 by unrelated DNA sequences does not change the cyclic AMP concentration dependency for in vitro transcription at P and inhibition of E2. Although the presence of some DNA to the left of -60 appears to be needed for efficient in vitro transcription at , the gal sequence to the left of -60 does not provide any specific interactions for transcription initiation at Pl. Similarly, efficient in vitro transcription from .2 also requires non-specific DNA sequences to the left of -60. We have also examined which bases were protected by RNA polymerase and CRP together from the action of DNAase and dimethylsulfate. Some of the interactions that take place when cAMP-CRP alone interacts with gal DNA appear to be preserved in the cAMP-CRP-RNA polymerase-gal DNA complex, suggesting that CRP occupies the same site in the DNA when it is alone or together with RNA polymerase. Our results suggest that the formation of an open complex at different promoters can result from different interaction patterns between RNA polymerase and promoter DNA.

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“…CAP has been shown to interact cooperatively with RNA polymerase at the lac and gal promoters (18,23,(28)(29)(30) and with the lac repressor at the lac promoter (15). In addition to these heterologous interactions, CAP interacts cooperatively with itself when it binds specifically to the gal promoter (28) in the presence of cAMP and when it binds nonspecifically to DNA in the absence of cAMP (13,25,32 10 mM MgCI2, and 100 mM KCl. The -2-fold increase in K11K2 accompanying the addition of 50 mM potassium glutamate to the reaction buffer indicates that K1 and K2 have different salt dependencies.…”

Section: Discussionmentioning

confidence: 99%

The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro

Hudson

Fried

1991

J Bacteriol

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The lactose promoter-operator region of Escherichia coli contains two binding sites for cyclic AMP receptor protein (CAP), two for the lactose repressor, and two for RNA polymerase. The high density of binding sites makes cooperative interactions between these proteins likely. In this study, we used the gel electrophoresis mobility shift assay and binding partition analysis techniques to determine whether the secondary CAP site influences the binding of CAP to the principal CAP site in the lactose promoter when both are present on a linear DNA molecule. Such an effect could occur through the formation of a bridged DNA-CAP-DNA structure, through the interaction of CAP molecules bound to each of the sites, or through allosteric effects caused by CAP-mediated DNA bending. We found, however, that the interaction of CAP with these sites was not cooperative, indicating that CAP sites 1 and 2 bind CAP in an independent manner.

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Two catabolite activator protein molecules bind to the galactose promoter region of Escherichia coli in the presence of RNA polymerase

Abstract: The catabolite activator protein (CAP) of Escherichia coli, complexed with cAMP, is required for efficient initiation of transcription from the galactose P1 promoter (start site at + 1) but not from the overlapping P2 promoter (start site at -5) [

Cited by 44 publications

References 27 publications

Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein

Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein

Interactions of RNA polymerase and the cyclic AMP receptor protein on DNA of theE. coligalactose operon

The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro

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