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

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 ϳ12 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.Transcription initiation requires formation of a preinitiation complex (PIC), 1 melting of DNA, formation of the first phosphodiester bonds, and promoter clearance involving movement of the open DNA region ("transcription bubble") and RNA polymerase. Finally, a stable ternary elongation complex is formed. These steps have been extensively studied during the last 2 decades in bacterial RNA polymerase and eukaryotic polymerase II (for reviews see Refs. 1 and 2) and to less extent in eukaryotic RNA polymerase III (3, 4) and RNA polymerase I (5) systems. In Archaea, open complex formation at the Methanococcus tRNA Val (6) and at the 16 S rRNA promoter of Sulfolobus (7) have been studied. The transition from initiation to elongation has not yet been investigated in Archaea.In bacteria, promoter isomerization from closed to open complex catalyzed by the predominant RNA polymerase holoenzyme (␤␤Ј␣ 2 70 ) occurs spontaneously in a temperature-dependent manner (8, 9). By contrast, nuclear RNA polymerase II (pol II; see Ref. 10) and Escherichia coli RNA polymerase specific for promoters of genes involved in nitrogen metabolism (␤␤Ј␣ 2 54 ; see Ref. 11) require ATP hydrolysis for promoter melting. In the pol II system promoter opening involves the helicase activity of TFIIH (12). Eukaryotic nuclear RNA polymerases I and III share with the 70 containing E. coli RNA polymerase the ability to produce an open complex of ...

The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation

“…DNA templates were attached to magnetic beads via the biotin-labeled 5Ј-end of the coding DNA strand as described previously (23). A footprinting reaction contained 168 fmol of DNA template, 70 nM purified RNAP or 70 nM RNAP⌬EЈF, 285 nM TBP, and 60 nM TFB in 25 l of transcription buffer not containing ␤-mercaptoethanol.…”

“…The samples were incubated for 5 min at 60 or 70°C as indicated. The reaction was stopped and exposed to piperidin treatment as described previously (23).…”

“…Complexes-Biotinylated C-minus cassettes of the gdh promoter attached to streptavidin-coated magnetic beads were used as DNA templates for stalling of transcription complexes at position ϩ20 (23 (23) and all washing and resuspending steps were carried out with preheated (60 -70°C) transcription buffer. Standard immobilized transcription reactions were incubated for 30 min at 60 or 70°C and stopped by the addition of loading dye as described (23) followed by denaturation for 3 min at 95°C.…”

“…Standard immobilized transcription reactions were incubated for 30 min at 60 or 70°C and stopped by the addition of loading dye as described (23) followed by denaturation for 3 min at 95°C.…”

“…We are exploring the mechanism and regulation of transcription in the hyperthermophilic Archaea Pyrococcus furiosus (23)(24)(25)(26)(27). In this contribution we report the reconstitution of a highly active RNAP from 11 bacterially expressed subunits.…”