Abstract
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, whereas 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 using 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 2 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.
Highlights
The recent past has seen a number of structures of RNA polymerases, ranging from RNA polymerase II (1, 2) to a smaller bacterial RNA polymerase (3) to the simplest well studied system: the single-subunit T7 RNA polymerase (4 –7)
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, whereas recent fluorescence and biochemical studies paint a preliminary picture of an elongation complex
Footprinting studies have indicated that complexes stalled at position ϩ6 retain promoter occupancy, as in the crystal structure, but complexes stalled at position ϩ15 have cleared the promoter (9, 12)
Summary
The recent past has seen a number of structures of RNA polymerases, ranging from RNA polymerase II (1, 2) to a smaller bacterial RNA polymerase (3) to the simplest well studied system: the single-subunit T7 RNA polymerase (4 –7). Lacking in any system is x-ray structural information on the critical transition from an unstable initially transcribing complex (ITC) to a stable elongation complex This transition occurs at about 10 base pairs (bp) in all RNA polymerases, suggesting that the transition is a fundamental feature of transcription, independent of the specific system (8). We have previously used fluorescent base analogs to map DNA melting in both the initial promoter-bound complex and in a stably elongating complex (beyond the transition) in the model enzyme T7 RNA polymerase (11, 13). The details of the model for the initial melted complex derived from the early fluorescence study have been confirmed by recent crystal structures (6), and the results from the latter are consistent with biochemical probes of a stalled elongation complex (9). The results present a detailed structural picture of promoter clearance in the T7 RNA polymerase model system
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