Structure-function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding and RNA displacement.

The active center clefts of RNA polymerase (RNAP) from the archaeon Pyrococcus furiosus (Pfu) and of yeast RNAP II are nearly identical, including four protruding loops, the lid, rudder, fork 1 and fork 2. Here we present a structure-function analysis of recombinant Pfu RNAP variants lacking these cleft loops, and analyze the function of each loop at different stages of the transcription cycle. All cleft loops except fork 1 were required for promoter-directed transcription and efficient elongation. Unprimed de novo transcription required fork 2, the lid was necessary for primed initial transcription. Analysis of templates containing a pre-melted bubble showed that rewinding of upstream DNA drives RNA separation from the template. During elongation, downstream DNA strand separation required template strand binding to an invariant arginine in switch 2, and apparently interaction of an invariant arginine in fork 2 with the non-template strand.

A polymerase III-like reinitiation mechanism is operating in regulation of histone expression in archaea.

An archaeal histone gene from the hyperthermophile Pyrococcus furiosus containing four consecutive putative oligo-dT terminator sequences was used as a model system to investigate termination signals and the mechanism of termination in vitro. The archaeal RNA polymerase terminated with high efficiency at the first terminator at 90 degrees C when it contained five to six T residues, at 80 degrees C readthrough was significantly increased. A putative hairpin structure upstream of the first terminator had no effect on termination efficiency. Template competition experiments starting with RNA polymerase molecules engaged in ternary complexes revealed recycling of RNA polymerase from the terminator to the promoter of the same template. This facilitated reinitiation was dependent upon the presence of a terminator sequence suggesting that pausing at the terminator is required for recycling as in the RNA polymerase III system. Replacement of the sequences immediately downstream of the oligo-dT terminator by an AT-rich segment improved termination efficiency. Both AT-rich and GC-rich downstream sequences seemed to impair the facilitated reinitiation pathway. Our data suggest that recycling is dependent on a subtle interplay of pausing of RNA polymerase at the terminator and RNA polymerase translocation beyond the oligo-dT termination signal that is dramatically affected by downstream sequences.

Exonuclease III footprinting on immobilized DNA templates.

DNA footprinting is a widely used method to locate the binding sites of protein on the DNA. It is based on the observation that a protein bound to DNA protects it from degradation by an enzyme or chemical reagent.Exonuclease III is a suitable probe to analyze the boundaries of a protein when it is necessary to eliminate any excess unbound DNA from the reaction to avoid background problems. In combination with biotin-labeled DNA that is bound to streptavidin-coated magnetic particles, information on the precise position of a DNA bound protein is available within a few hours. The position of the archaeal RNA polymerase at different stages of transcription in the Pyrococcus furiosus in vitro transcription system was analyzed by this method.

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 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.