Promoter Clearance by T7 RNA Polymerase: Initial Bubble Collapse and Transcript Dissociation Monitored by Base Analog Fluorescence
Footprinting, fluorescence and x-ray structural information from the initial, promoter bound complex of T7 RNA polymerase describes the very beginning of the initiation of transcription, while recent fluorescence and biochemical studies paint a preliminary picture of an elongation complex. The current work focuses on the transition from an initially transcribing, promoter bound complex, to an elongation complex clear of the promoter. Fluorescence quenching is used to follow the melted state of the DNA bubble, and a novel approach employing a locally mismatched fluorescent base analog reports on the local structure of the heteroduplex. Fluorescent base analogs placed at positions ?2 and ?1 of the promoter indicate that this initially melted, nontranscribed region remains melted as the polymerase translocates through to position +8. In progressing to position +9, this region of the DNA bubble begins to collapse. Probes placed at positions +1 and +2 of the template strand indicate that the 5′ end of the RNA remains in a heteroduplex as the complex translocates to position +10. Subsequent translocation leads to sequential dissociation of the first two bases of the RNA. These results show that the initially transcribing complex bubble can reach a size of up to 13 base pairs, and a maximal heteroduplex length of 10 base pairs. They further indicate that initial bubble collapse precedes dissociation of the 5′ end of the RNA.