Structural kinetics of transcription activation at the malT promoter of Escherichia coli by UV laser footprinting.
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.
Eichenberger
P
P
Département de Biologie Moléculaire, Université de Genève, 30 Quai Ernest-Ansermet, CH-1211 Genève 4, Switzerland.
Déthiollaz
S
S
Buc
H
H
Geiselmann
J
J
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D007834:Lasers; D011401:Promoter Regions, Genetic / Q000235:genetics; D014157:Transcription Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058976:MalT protein, E coli; D014157:Transcription Factors