[Show abstract][Hide abstract] 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.
Full-text · Article · Sep 1997 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The interactions between the cAMP receptor protein (CRP) and RNA polymerase during transcriptional activation at the Escherichia coli malT promoter have been analyzed using a combination of footprinting methods. We show that a closed complex is formed at this promoter in the absence of activator and that CRP merely stabilizes the open complex. The alpha-subunits of the RNA polymerase are involved in this effect as shown by KMnO4 footprinting. The open complex formed in the presence of CRP is structurally identical to the one found at a CRP-independent promoter up-mutant. UV-laser footprinting yields distinct signals for the different protein-DNA interactions within the complex and for interactions between CRP and RNA polymerase. We monitor these signals in promoter variants that place the CRP binding site at different distances upstream of the start site of transcription. Signals within the core promoter region, as well as those located just upstream of the -35 hexamer, are unaffected by the position of the CRP binding site. Contacts of RNA polymerase with the upstream promoter region change in a mutant RNA polymerase containing a truncated alpha-subunit. We conclude that at least one of the alpha-subunits of RNA polymerase binds to DNA upstream of the -35 hexamer and that this interaction is unaffected by the position of the CRP binding site. We discuss models that account for the different activities of CRP in transcriptional activation as a function of promoter geometry.
[Show abstract][Hide abstract] ABSTRACT: Transcription from many Escherichia coli promoters can be activated by the cAMP-CRP complex bound at different locations upstream of the promoter. At some locations the mechanism of activation involves direct protein-protein contacts between CRP and the RNA polymerase. We positioned the CRP binding site at various distances from the transcription start site of the malT promoter and measured the in vivo activities of these promoter variants. From the activation profiles we deduce that the protein-protein interactions involved in transcriptional activation are rather rigid. A heterologous protein (IHF) that bends the DNA to a similar degree as does CRP activates transcription when bound at sites equivalent to activating positions for CRP. DNA geometry makes a major contribution to the process of transcriptional activation and DNA upstream of the activator binding site participates in this process. Removal of this DNA decreases the capacity of the malT promoter to be activated by CRP in vitro. We conclude that both DNA topology and direct protein-protein contacts contribute to transcriptional activation and that the relative importance of these two modes of activation depends on the nature of the activator and on the location of the activator binding site.