Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoter

Malan, T.Philip; Kolb, Annie; Buc, Henri; McClure, William R.

doi:10.1016/0022-2836(84)90262-6

Cited by 221 publications

(142 citation statements)

References 30 publications

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“…The inferred values of K B (1 ϫ 10 7 M Ϫ1 ) and k 2 (0.3 s Ϫ1 ) are consistent with expectation based on conventional experiments (14)(15)(16)(17). The fourth observable is the time interval between an unwinding event and the subsequent rewinding event (T unwound ; Figs.…”

Section: Resultssupporting

confidence: 90%

Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation

Revyakin

Ebright²,

Strick³

2004

Proc. Natl. Acad. Sci. U.S.A.

158

204

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By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP). By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with Ϸ1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with Ϸ5-nm resolution). We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides. We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules. We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils). The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and͞or DNA compaction.T ranscription initiation involves a series of reactions (1-2): (i) RNA polymerase holoenzyme (RNAP) binds to promoter DNA to form an RNAP-promoter closed complex; (ii) RNAP unwinds approximately one turn of the promoter DNA to form an RNAP-promoter open complex (in a process referred to as ''promoter unwinding''); and (iii) RNAP escapes the promoter and enters into productive synthesis of RNA as an RNAP-DNA elongation complex (in a process referred to as ''promoter clearance'').We have developed a single-molecule DNA-nanomanipulation approach that enables us to detect and characterize promoter unwinding and promoter clearance by RNAP. Our approach uses an experimental setup originally developed for analysis of DNA polymer physics . In this experimental setup, a double-stranded DNA molecule containing a single promoter site is attached at one end, through multiple linkages, to a paramagnetic bead, and at the other end, through multiple linkages, to a glass surface; the DNA is torsionally constrained and mechanically stretched between the bead and the glass surface by application of a pair of magnets above the DNA helix axis; and the distance between the bead and the glass surface (which reflects the DNA end-to-end extension, l) is monitored in real time by using videomicroscopy. Upon rotation of the pair of magnets, the bead is rotated in lock-step register, superhelical turns are introduced into the torsionally constrained DNA molecule in lock-step register, supercoils are formed, and, correspondingly, l is changed. With this experimental setup, it readily is possible to construct an experimental calibration curve relating l to the number of clockwise or counterclockwise rotations of the pair of magnets, and thus to the number of negative or positive superhelical turns ( Fig. 1B; refs. 3-5). Over a b...

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Section: Resultssupporting

confidence: 90%

Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation

Revyakin

Ebright²,

Strick³

2004

Proc. Natl. Acad. Sci. U.S.A.

158

204

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“…We and others have noted the striking similarities between the arrangement of signals in the lac and gal regulatory regions [11,19,20]: in both cases there are two overlapping promoters, P1 and P2, one of which is stimulated and the other of which is blocked by cAMP-CRP. This work suggests a third common feature, as the gal P3 promoter that initiates transcription at + 14/+ 15, is strikingly similar to the Pl15 promoter in the lac operon that initiates transcription at +13 [21].…”

Section: Discussionmentioning

confidence: 71%

“…However, from our studies in vitro we can draw two clear conclusions concerning the action of CRP. Firstly, it is generally thought that CRP stimulates transcription by acting at an early stage during the recognition of promoter DNA by RNA polymerase probably by directly contacting incoming polymerase [20,. The -10 hexamer sequence cannot be essential for this step as CRP acts even when the hexamer is altered from 5'-TATGGT-3' to 5 '-CATGGT-3'.…”

Section: Discussionmentioning

confidence: 99%

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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“…Indeed, all three of the pairwise binding interactions examined to date in this system have exhibited significant cooperativity. CAP bound to CAP site 1 prevents RNA polymerase binding to promoter P2 (19) and stabilizes polymerase binding to promoter P1 by 3.5-to 15-fold (18,30). Conversely, RNA polymerase has been shown to stabilize the lac promoter complexes of a class of CAP mutants that can activate transcription in vivo in the absence of cAMP (23).…”

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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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Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoter

Cited by 221 publications

References 30 publications

Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation

Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation

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

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

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