Transcription by an archaeal RNA polymerase is slowed but not blocked by an archaeal nucleosome

Abstract

Archaeal RNA polymerases (RNAPs) are closely related to eukaryotic RNAPs, and in Euryarchaea, genomic DNA is wrapped and compacted by histones into archaeal nucleosomes. In eukaryotes, transcription of DNA bound into nucleosomes is facilitated by histone tail modifications and chromatin remodeling complexes, but archaeal histones do not have histone tails and archaeal genome sequences provide no evidence for archaeal homologs of eukaryotic chromatin remodeling complexes. We have therefore investigated the ability of an archaeal RNAP, purified from Methanothermobacter thermautotrophicus, to transcribe DNA bound into an archaeal nucleosome by HMtA2, an archaeal histone from M. thermautotrophicus. To do so, we constructed a template that allows transcript elongation to be separated from transcription initiation, on which archaeal nucleosome assembly is positioned downstream from the site of transcription initiation. At 58 degrees C, in the absence of an archaeal nucleosome, M. thermautotrophicus RNAP transcribed this template DNA at a rate of approximately 20 nucleotides per second. With an archaeal nucleosome present, transcript elongation was slowed but not blocked, with transcription pausing at sites before and within the archaeal nucleosome. With additional HMtA2 binding, complexes were obtained that also incorporated the upstream regulatory region. This inhibited transcription presumably by preventing archaeal TATA-box binding protein, general transcription factor TFB, and RNAP access and thus inhibiting transcription initiation.

FIG. 1.

Transcription template and position of HMtA2 assembly. (A) The sequence of the transcription template is shown with the BRE and TATA-box sequence from the HMtB promoter (16), the U-less cassette (dark gray), Selex1 sequence (light gray) (4), site of transcription initiation (→), and 3′ (▾) and 5′ (asterisks) boundaries of the MN-protected fragments indicated. (B) Electrophoretic separation of the fragments of the template DNA protected from MN digestion by assembly into complexes at the HMtA2 dimer/100-bp ratios indicated above the corresponding lane. Control lanes contained size standards (S), untreated template DNA (−) and template DNA exposed to MN for 1 min in the absence of HMtA2 (0). (C) Autoradiogram of the electrophoretic separation of the restriction fragments generated from a population of the ∼90-bp MN-protected DNA fragments after ³²P end labeling and digestion with EcoRI (E), BamHI (B), or NruI (N). Control lanes contained size standards (S) and an aliquot of the ∼90-bp molecules not exposed to restriction enzymes (−). (D) Diagram showing the position of the archaeal nucleosome (shaded oval), the 5′ and 3′ boundaries, and the precise lengths of the fragments of the template DNA protected from MN digestion as determined by the lengths of the primer extension products indicated below. The extension products were generated from ³²P-labeled primers that hybridized to the MN-protected fragments bound by 5, 15, or 30 HMtA2 dimers per 100 bp of template DNA. Adjacent lanes contained the products of sequencing reactions (G, C, T, A) generated from the template DNA, using the same primers.

FIG. 2.

Runoff transcription and EMSA of HMtA2 binding. (A) Electrophoresis of the 225-nt ³²P-labeled runoff transcripts synthesized in 30 min at 58°C on templates preincubated with HMtA2 as indicated (dimers per 100 bp) above each lane. The amount of transcript, as a percentage of that synthesized in the absence of HMtA2 (−) is listed below each lane. Lane S contained size standards. (B) Autoradiogram of the electrophoretic separation of the complexes formed by incubation of ³²P-labeled template DNA (50 ng) without (−) and with HMtA2 at the histone dimer/100-bp ratio indicated above each lane.

FIG. 3.

Stalled-transcript elongation and EMSA of ternary complexes. (A) Ternary complexes that contained the ³²P-labeled 24-nt U-less transcript were incubated without (−) or with HMtA2 and then added to a complete transcription reaction mixture and incubated at 58°C for 20 min. A control aliquot of the ternary complexes was incubated in a reaction mixture that lacked UTP (−UTP). The transcripts synthesized were separated by electrophoresis, and the ³²P-labeled transcripts were detected by autoradiography and quantitated by β-decay measurement. The HMtA2 dimer/100-bp ratio in the reaction mixture is indicated above the corresponding lane. The control lanes contained size standards (S1 and S2). (B) Aliquots of ternary complexes that contained a ³²P-labeled 24-nt U-less transcript were incubated with HMtA2 at the histone dimer/100-bp ratio indicated above each lane. The products were separated by electrophoresis and visualized by autoradiography. Control lanes contained a sample of ³²P-end-labeled template DNA (T), an aliquot of the ternary complexes incubated without HMtA2 addition (−), and an aliquot incubated with the HMfB K13T+R19S+T54K variant which lacks DNA binding ability (51) at a ratio of 30 histone dimers per 100 bp (C).

FIG. 4.

Transcription in the absence and presence of an archaeal nucleosome. Ternary complexes containing a ³²P-labeled 24-nt U-less transcript incubated with HMtA2 (+HMtA2) or without HMtA2 (−HMtA2) were added to a complete reaction mixture and placed at 58°C. Aliquots were taken at the times indicated (seconds), and the transcripts synthesized were separated by electrophoresis and visualized by phosphorimaging. Control lanes contained nucleotide size standards (S1 and S2). In the diagrams, the template DNA is shown to scale, with or without the positioned archaeal nucleosome. Footprinting studies indicate that an archaeal RNAP in a ternary complex extends ∼12 bp downstream from the site of nucleoside triphosphate polymerization (52). The rate of transcription was estimated, as indicated, from the increase in length of transcripts during a 2-s incubation at 58°C.