Structural kinetics of transcription activation at the malT promoter of Escherichia coli by UV laser footprinting - PubMed (original) (raw)
Structural kinetics of transcription activation at the malT promoter of Escherichia coli by UV laser footprinting
P Eichenberger et al. Proc Natl Acad Sci U S A. 1997.
Abstract
We have studied the kinetics of transcriptional initiation and activation at the malT and malTp1 promoters of Escherichia coli using UV laser footprinting. Contrary to previous studies and because of the very rapid signal acquisition by this technique, we can obtain structural information about true reaction intermediates of transcription initiation. The consequences of adding a transcriptional activator, the cAMP receptor protein/cAMP complex (CRP), are monitored in real time, permitting us to assign specific interactions to the activation of discrete steps in transcription initiation. Direct protein-protein contacts between CRP and the RNA polymerase appeared very rapidly, followed by DNA melting around the -10 hexamer. CRP slightly increased the rate of this isomerization reaction but, more importantly, favored the establishment of additional contacts between the DNA upstream of the CRP binding site and RNA polymerase subsequent to open complex formation. These contacts make a major contribution to transcriptional activation by stabilizing open forms of the promoter complex, thereby indirectly accelerating promoter escape. The ensemble of the kinetic, structural signals demonstrated directly that CRP exerts most of its activating effects on the late stages of transcriptional initiation at the malT promoter.
Figures
Figure 1
Kinetics of open complex formation at the malTp1 promoter in the presence of CRP. (a) UV laser footprints of the core promoter region. The samples at t = 0 min have been irradiated before addition of RNA polymerase. For the other samples, the time shown above the lanes indicates the incubation time before UV irradiation. The intensity of the band at −9 correlates with DNA melting (open complex formation). (b) UV laser footprints of the upstream promoter region. The band at −94 characterizes the interaction of upstream DNA with RNA polymerase; the band at −60 decreases in intensity upon interaction between CRP and RNA polymerase. (c) Time course of open complex formation at the malTp1 promoter. The normalized band intensity is plotted as a function of time. Filled circles, band at −9, +CRP; open circles, band at −9, no CRP; squares, band at −94, +CRP.
Figure 2
Time course of open complex formation at the malT promoter in the presence of CRP. Circles, band at −9; Squares, band at −94.
Figure 3
KMnO4 footprints of transcription complexes at the malT promoter in the presence of limited sets of NTPs. ATP is present in lanes 3 and 9, ATP + UTP are present in lanes 4 and 10, ATP + UTP + CTP are present in lanes 5 and 11, and all four NTPs are present in lanes 6 and 12. CRP is present in lanes 7–12. A linearized plasmid is used for normalization, and the corresponding band is indicated with an asterisk.
Figure 4
Kinetics of transcription initiation at the malT promoter in the presence of three NTPs. (a) UV laser footprints in the presence of CRP. The bands at +1 and +12 are characteristic of the complex halted at +9 by deprivation of GTP. (b) Time course of formation of the transcription initiation complex. The normalized intensity of the band at +12 is plotted as a function of the time after addition of RNA polymerase. The absolute band intensities of the samples lacking CRP are three times smaller than the corresponding intensities in the presence of CRP. Filled triangles, + CRP; open triangles, no CRP.
Figure 5
(a) Model of transcriptional activation by CRP at the malT promoter. The CRP dimer is symbolized as a two-domain ellipsoid. The DNA (solid line) is bent around the prebound CRP. Newly appearing signals visible in UV laser footprinting experiments are indicated with an asterisk. The reaction is divided into three steps. (i) RNA polymerase (large ellipsoid) makes a direct contact with the prebound CRP via the carboxyl-terminal domain of the α-subunits (represented by the darker ellipsoids). Signals appear at −32 and at −60. (ii) RNA polymerase opens the DNA between the −10 hexamer and the start site of transcription. (iii) Contacts between the upstream DNA and the back of RNA polymerase stabilize the open region of DNA. The symmetry properties of the α-subunits are arbitrary; the interaction of both α-subunits with the DNA may be restricted to the stretch just upstream of the promoter. (b) Change in free energy during transcription initiation and activation. The solid line represents the malT promoter and the stippled line represents the same promoter in the presence of CRP. The diagram illustrates the redistribution of the most populated state (represented by the circles) in the presence of CRP. The rate-limiting step (highest peak) remains promoter escape under both conditions, and open complex formation is slightly accelerated in the presence of CRP. The diagram is schematic to illustrate how CRP indirectly accelerates promoter escape, and it is not meant to accurately reflect absolute free energies.
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