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J. Biol. Chem., Vol. 278, Issue 33, 30497-30505, August 15, 2003

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Analysis of the Open Region and of DNA-Protein Contacts of Archaeal RNA Polymerase Transcription Complexes during Transition from Initiation to Elongation^*

Patrizia Spitalny  and Michael Thomm  

From the Universität Kiel, Institut für Allgemeine Mikrobiologie, Am Botanischen Garten 1-9, D-24118 Kiel, Germany

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 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.

Received for publication, April 8, 2003 , and in revised form, May 26, 2003.

^* This work was supported by a grant from the Deutsche Forschungsgemeinschaft and of the Fonds der Chemischen Industrie (to M. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Universität Regensburg, Lehrstuhl für Mikrobiologie, Universitätsstr. 31, D-93053 Regensburg, Germany.

To whom correspondence should be addressed. Tel.: 49-941-943-3160; Fax: 49-941-943-2403; E-mail: Michael.Thomm{at}Biologie.Uni-Regensburg.de.

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