Mechanism of activation of transcription by the complex formed between cyclic AMP and its receptor in Escherichia coli.

and single-strand-specific cleavage. The two open complexes produce identical deoxyribonuclease I footprints and similar methylation interference/protection (one base shows different behaviour), indicating that both complexes are at the same binding site. The two open complexes, however, differ in the amount of unwinding in the 13 to + 3 region, as assayed by probes for singlestranded DNA. A functional assay, transcription within the gel slices containing the open complexes, indicates that the open complexes also differ in the ability to escape from the abortive initiation cycle into productive transcription. The upper gel band 0, has a lower total RNA synthesis rate but a greater relative production of the stably bound 1 1-mer compared with the abortive 8-mer product. Dominance of 0, at higher temperature may reflect an adaptive mechanism to counter the observed tendency of abortive initiation to increase with increasing temperature. The results are summarized in the scheme shown in Fig. 1. Deoxyribonuclease I footprinting of the initiated complex containing only the first stable transcript (the 1 1-mer) shows a contraction from the open-complex footprints by loss of approximately 25 bp of contacts in the 35 region, and loss of the o-subunit. This drastic structural change, and the measured apparent activation energy for abortive release of nascent RNA transcripts, lead us to propose a model which features an energetically stressed intermediate in the translocation of polymerase away from its contact in the 35 region. The model focuses on mutually competitive interactions of polymerase with the 35 region or with short transcripts in the 0 to + 10 region; the stability of the RNA-DNA hybrid double helix formed by the pascent transcripts can also be expected to affect the relative importance of abortive initiation.