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 activat ion at the malT and malTp1 promoters of Escherichia coli using UV lase r footprinting. Contrary to previous studies and because of the very r apid signal acquisition by this technique, we can obtain structural in formation about true reaction intermediates of transcription initiatio n. The consequences of adding a transcriptional activator, the cAMP re ceptor protein/cAMP complex (CRP), are monitored in real time, permitt ing us to assign specific interactions to the activation of discrete s teps in transcription initiation, Direct protein-protein contacts betw een CRP and the RNA polymerase appeared very rapidly, followed by DNA melting around the -10 hexamer. CRP slightly increased the rate of thi s isomerization reaction but, more importantly, favored the establishm ent of additional contacts between the DNA upstream of the CRP binding sire and RNA polymerase subsequent to open complex formation. These c ontacts make a major contribution to transcriptional activation by sta bilizing open forms of the promoter complex, thereby indirectly accele rating promoter escape, The ensemble of the kinetic, structural signal s demonstrated directly that CRP exerts most of its activating effects on the late stages of transcriptional initiation at the malT promoter .