Abstract
The archaeal transcriptional machinery is polymerase II (pol II)-like but does not require ATP or TFIIH for open complex formation. We have used enzymatic and chemical probes to follow the movement of Pyrococcus RNA polymerase (RNAP) along the glutamate dehydrogenase gene during transcription initiation and transition to elongation. RNAP was stalled between registers +5 and +20 using C-minus cassettes. The upstream edge of RNAP was in close contact with the archaeal transcription factors TATA box-binding protein/transcription factor B in complexes stalled at position +5. Movement of the downstream edge of the RNAP was not detected by exonuclease III footprinting until register +8. A first structural transition characterized by movement of the upstream edge of RNAP was observed at registers +6/+7. A major transition was observed at registers +10/+11. In complexes stalled at these positions also the downstream edge of RNA polymerase started translocation, and reclosure of the initially open complex occurred indicating promoter clearance. Between registers +11 and +20 both RNAP and transcription bubble moved synchronously with RNA synthesis. The distance of the catalytic center to the front edge of the exo III footprint was approximately 12 nucleotides in all registers. The size of the RNA-DNA hybrid in an early archaeal elongation complex was estimated between 9 and 12 nucleotides. For complexes stalled between positions +10 and +20 the size of the transcription bubble was around 17 nucleotides. This study shows characteristic mechanistic properties of the archaeal system and also similarities to prokaryotic RNAP and pol II.
Highlights
Last 2 decades in bacterial RNA polymerase and eukaryotic polymerase II and to less extent in eukaryotic RNA polymerase III [3, 4] and RNA polymerase I [5] systems
The upstream edge of RNA polymerase (RNAP) was in close contact with the archaeal transcription factors TATA box-binding protein/transcription factor B in complexes stalled at position ؉5
We have developed recently a cell-free transcription system for the hyperthermophile Pyrococcus furiosus [26]. This highly purified system consisting of bacterially produced TATA box-binding protein (TBP) and transcription factor B (TFB) and RNA polymerase isolated from Pyrococcus cells was used for the characterization of the archaeal preinitiation complex [27], analysis of the trajectory of DNA in an archaeal transcription complex [28], and first studies on regulation of transcription in Archaea [29, 30]
Summary
Reagents and Enzymes—Exonuclease III was purchased from New England Biolabs. Potassium permanganate was obtained from Merck. [␣-32P]UTP and [␥-32P]ATP were purchased from Hartmann Bioanalytics (Braunschweig, Germany). Exonuclease III Footprinting—To perform footprinting experiments, the immobilized DNA templates were labeled with [␥-32P]ATP on the free 5Ј-end of either the coding or the RNA-like strand, depending on which strand was attached to the magnetic particle on the 5Ј-end. After the complexes had been stalled at positions ϩ5 to ϩ11, ϩ15, and ϩ20 relative to the transcription start site, they were isolated as described They were resuspended in 25 l of reaction buffer for exo III digestion (40 mM KCl, 2 mM MgCl2, 100 mM Tris-HCl, pH 8.5, and 1 mM dithiothreitol). To detect the open complex 2.5 l of KMnO4 (250 mM) were added immediately after incubation of template DNA with TBP, TFB, and RNA polymerase for 3 min at 70 °C, and the reaction was performed for another 3 min at 70 °C. The reaction was stopped and subjected to piperidine treatment as described
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