Events during initiation of archaeal transcription: open complex formation and DNA-protein interactions

Abstract

Transcription in Archaea is initiated by association of a TATA box binding protein (TBP) with a TATA box. This interaction is stabilized by the binding of the transcription factor IIB (TFIIB) orthologue TFB. We show here that the RNA polymerase of the archaeon Methanococcus, in contrast to polymerase II, does not require hydrolysis of the beta-gamma bond of ATP for initiation of transcription and open complex formation on linearized DNA. Permanganate probing revealed that the archaeal open complex spanned at least the DNA region from -11 to -1 at a tRNA(Val) promoter. The Methanococcus TBP-TFB promoter complex protected the DNA region from -40 to -14 on the noncoding DNA strand and the DNA segment from -36 to -17 on the coding DNA strand from DNase I digestion. This DNase I footprint was extended only to the downstream end by the addition of the RNA polymerase to position +17 on the noncoding strand and to position +13 on the coding DNA strand.

FIG. 1

Transcription in the absence of ATP. (A) Schematic drawing of the template pIC-31/2. The nucleotide sequence of the promoter region and the region around the transcription start site (+1) is shown. In the absence of ATP, synthesis of a 6-nt RNA product (+6) was expected. (B) Transcription reaction mixtures contained RNA polymerase, 4 μM UTP, 20 μM CTP, and template pIC-31/2. The presence (+) or absence (−) of aTBP, TFB, and GTP (20 μM) in the reaction mixture is indicated. For size calibration of the RNA product, the ATP analogue 3′-dATP was added to allow incorporation of the next nucleotide (lane C).

FIG. 2

Transcription in the presence of GTP analogs and in the presence of the dinucleotide UpG. Transcription reactions were performed as described for Fig. 1 and in Materials and Methods. (A) GTP (lanes 1 to 3) was replaced by 80 μM GTPγS (lanes 4 to 6) and 80 μM GMP-PNP (lanes 7 to 9). Addition of dGTP (20 μM) and dATP (20 μM) is indicated on top of the gel. The expected 6-nt RNA product is marked by an arrow. (B) The presence (+) or absence (−) of aTBP, TFB, and UpG (170 μM) in the reaction mixture is indicated. Note that the electrophoretic mobility of the dinucleotide-initiated transcript (labeled by an arrow) was reduced compared to that of the GTP-initiated transcript (lane C).

FIG. 3

Open complex formation. (A) Transcription reaction mixtures for open complex formation were assembled as described in Materials and Methods. Linearized pIC-31/2 wild-type DNA was incubated with the components indicated on top of each lane for 20 min at 55°C. After treatment with KMnO₄, the hyperreactive sites on the nontemplate (lanes 1 to 6) and the template strand (lanes 6′ to 11) were analyzed by asymmetric PCR using radioactive labeled primers. Positions of reactive thymidine residues were determined by comigration of a sequence ladder terminated with ddATP and are indicated in relation to the transcription start site. Compare the following: lanes 1 and 6′, without protein and nucleotides as a control; lanes 2, 3, 7, and 8, without nucleotides; lanes 4 to 6 and 9 to 11, in the presence of 20 μM ATP (A), GTP (G), or CTP (C). (B) DNA sequence of plasmid pIC-31/2 containing the promoter and the region of the transcription start site. The positions of the modified thymidine residues are boxed. Position numbers refer to the transcription start site.

FIG. 4

Open complex formation and transcription at low temperatures. Transcription reactions for open complex formation were performed as described in Materials and Methods. Linearized (A) or negatively supercoiled (B) plasmid pIC-31/30PRO-C25 was incubated with the components indicated at the bottom of each panel for 20 min at the temperatures indicated. After treatment with KMnO₄, the hyperreactive sites on the nontemplate strand were analyzed by asymmetric PCR using a fluorescent-dye-labeled primer. For fragment size calibration of the peaks, size markers with a different fluorescent dye were added to each probe and size correlation was done by using the global Southern method according to the instructions of the supplier. The DNA sequences of the plasmid pIC-31/30PRO-C25 are shown on top in such a way that the peaks of the chromatograms can be directly correlated to the individual base positions within the DNA sequence. The transcription start site is underlined. (C) Transcription reaction mixtures were incubated for 20 min at the temperatures indicated on top of the lanes using negatively supercoiled (C) or linearized (L) DNA.

FIG. 5

Interaction of the transcriptional components with the tRNA^Val gene. The DNA fragment was incubated with the components (25 pmol of aTBP, 8 pmol of TFB, 0.8 pmol of RNA polymerase) as indicated at the bottom of each chromatogram for 20 min at 37°C, followed by DNase I treatment. Further treatment was as described in Materials and Methods. For size calibration of the peaks of the template strand, size markers with a different fluorescent dye were added to each probe and size calling was done by using the global Southern method according to the instructions of the supplier. The calculation of the fragment length was calibrated using sequencing reactions generated with the fluorescence-labeled primer. For the analysis of the nontemplate strand, the 220-bp DNA fragment was also generated by PCR using a biotinylated M13 reverse primer and a fluorescent-dye (ABI JOE)-labeled M13 primer. The DNA sequences of the plasmid pIC-31/30PRO-C25 are shown on the tops of the chromatograms in such a way that the peaks of the chromatograms can be directly correlated to the individual base positions within the DNA sequence. The promoter and the transcription start site are underlined. *, DNase I hypersensitive sites.

FIG. 6

Summary of the results of the DNase I footprinting experiments. The DNA sequence from −40 to +40 relative to the transcription start site is shown. The promoter and the transcription start site are boxed. The regions protected from DNase I digestion of the nontemplate and the template strand are shown as shaded rectangles below each sequence. The sequence of the TATA box is boxed and the sequence of the BRE element is hatched. DNase I hypersensitivity sites are labeled with lowercase letters.