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

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 initia...

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Bibliographic Details
Published in:The Journal of biological chemistry 2003-08, Vol.278 (33), p.30497-30505
Main Authors: Spitalny, Patrizia, Thomm, Michael
Format: Article
Language:English
Subjects:
Archaeal Proteins - genetics
Archaeal Proteins - metabolism
Base Sequence
DNA Footprinting
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
DNA-Directed RNA Polymerases - genetics
DNA-Directed RNA Polymerases - metabolism
Exodeoxyribonucleases
Molecular Sequence Data
Peptide Chain Elongation, Translational - physiology
Potassium Permanganate
Pyrococcus - enzymology
Pyrococcus - genetics
RNA, Archaeal - genetics
Transcription Initiation Site - physiology
Transcription, Genetic - genetics
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M303633200