Influence of the location of the cAMP receptor protein binding site on the geometry of a transcriptional activation complex in Escherichia coli.
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.
Eichenberger
P
P
Département de Biologie Moléculaire, Université de Genève, Switzerland.
Déthiollaz
S
S
Fujita
N
N
Ishihama
A
A
Geiselmann
J
J
D001426:Bacterial Proteins / Q000235:genetics; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D007834:Lasers; D008957:Models, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011953:Receptors, Cyclic AMP / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058976:MalT protein, E coli; D011953:Receptors, Cyclic AMP; D014157:Transcription Factors; D004247:DNA; D012321:DNA-Directed RNA Polymerases