Temperature, template topology, and factor requirements of archaeal transcription

Abstract

Although Archaea are prokaryotic and resemble Bacteria morphologically, their transcription apparatus is remarkably similar to those of eukaryotic cell nuclei. Because some Archaea exist in environments with temperatures of around 100 degreesC, they are likely to have evolved unique strategies for transcriptional control. Here, we investigate the effects of temperature and DNA template topology in a thermophilic archaeal transcription system. Significantly, and in marked contrast with characterized eucaryal systems, archaeal DNA template topology has negligible effect on transcription levels at physiological temperatures using highly purified polymerase and recombinant transcription factors. Furthermore, archaeal transcription does not require hydrolysis of the beta-gamma phosphoanhydride bond of ATP. However, at lower temperatures, negatively supercoiled templates are transcribed more highly than those that are positively supercoiled. Notably, the block to transcription on positively supercoiled templates at lowered temperatures is at the level of polymerase binding and promoter opening. These data imply that Archaea do not possess a functional homologue of transcription factor TFIIH, and that for the promoters studied, transcription is mediated by TATA box-binding protein, transcription factor TFB, and RNA polymerase alone. Furthermore, they suggest that the reduction of plasmid linking number by hyperthermophilic Archaea in vivo in response to cold shock is a mechanism to maintain gene expression under these adverse circumstances.

Figure 1

Effect of template topology and temperature on in vitro transcription from the S. shibatae 16S rRNA promoter by using a reconstituted (a) or unfractionated (10 μg of Sulfolobus crude cell extract) system (b). Transcription of negatively supercoiled template (lanes 1 and 3) or linearized template (lanes 2 and 4) was as described in Materials and Methods. Transcription reactions were incubated at 75°C (lanes 1 and 2) or 48°C (lanes 3 and 4) and RNA detected by primer extension.

Figure 2

Generation of topoisomer pools for use in the reconstituted archaeal transcription system. Negative image of ethidium bromide-stained two-dimensional gels resolving topoisomers of 16S promoter plasmid.

Figure 3

Transcription reactions using discrete topoisomer pools in the reconstituted Sulfolobus system. Transcription reactions using T6 topoisomer pools, at 75°C (a) or 48°C (e). The specific linking difference (σ) values, calculated as described in Materials and Methods, are given under the appropriate lanes. (b and f) Transcription reactions using 16S pools at 75°C and 48°C as indicated. (c and d) Effect of substituting adenosine 5′-[β,γ-imido]triphosphate (AMP-PNP) for ATP in the reconstituted transcription reaction on negatively supercoiled (σ = −0.042) T6 template (c) and positively supercoiled (σ = +0.056) T6 template (d) at 75°C. Lanes 1–3 contain 100, 50, and 25 μM ATP, respectively, together with 100 μM GTP, CTP, and UTP. Lanes 4–6 contain 100, 50, and 25 μM AMP-PNP, respectively, together with 100 μM GTP, CTP, and UTP.

Figure 4

DNaseI footprinting of archaeal TBP and TFB on the template strand of negatively supercoiled (σ = −0.053) 16S (lanes 1–7) and positively supercoiled (σ = +0.047) 16S templates (lanes 8–13). Lane 1 contains a ddA sequencing ladder. Reactions contain 0 pmol (lanes 2 and 8), 3 pmol (lanes 3 and 9), 1.5 pmol (lanes 4 and 10), 0.75 pmol (lanes 5 and 11), 0.38 pmol (lanes 6 and 12), and 0.19 pmol (lanes 7 and 13) of an equimolar mix of TBP and TFB. The region protected from cleavage by DNaseI is indicated by an open box; the TATA and initiator elements are shown as filled boxes.

Figure 5

Single-round transcription assays. (a) Establishment of single-round assay. Transcription of negatively supercoiled 16S promoter was conducted at 75°C with various orders of addition of heparin and NTPs, as indicated. (b) Linear (lanes 1, 3, 5, and 7) or supercoiled (lanes 2, 4, 6, and 8) template was preincubated with TBP, TFB, and RNAP in the presence of ATP, CTP, and GTP at 48°C (lanes 1–4) or 75°C (lanes 5–8). Heparin was added to prevent reinitiation, and reactions were incubated at 48°C (lanes 1, 2, 5, and 6) or 75°C (lanes 3, 4, 7, and 8) for an additional 5 min.

Figure 6

Analysis of promoter opening by permanganate probing of the 16S promoter. (a) Open complex formation on negatively supercoiled 16S(σ = −0.053) promoter at 48°C. Lanes 1 and 7 and lanes 2 and 8 contain ddG and ddA sequence ladders, respectively. Lanes contain modification pattern in the absence of TBP, TFB, and RNAP (−, lanes 3 and 9), modification with TBP, TFB, RNAP, and no nucleoside triphosphates (+, lanes 4 and 10), modification with TBP, TFB, RNAP, and GTP (+, GTP; lanes 5 and 11), and modification with protein with GTP and CTP (+, G, CTP; lanes 6 and 12). Modification is detected on both template (lanes 1–6) and nontemplate strand (lanes 7–12). The region of modified thymidines is indicated by a vertical bar on the right. (b) No open complex is detectable on positively supercoiled templates at 48°C. Permanganate modification assays were performed on negatively supercoiled 16S (σ = −0.053, lanes 1, 3, 5, and 7) or positively supercoiled 16S (σ = +.047, lanes 2, 4, 6, and 8) DNA at 48°C in the presence (lanes 3, 4, 7, and 8) or absence (lanes 1, 2, 5, and 6) of TBP, TFB, and RNAP. (c) Summary of permanganate sensitivity data. The sequence of the 16S promoter is shown with the position of modified Ts indicated by bold type and ∗. The TATA element is boxed, and the site of transcription initiation is indicated by an arrow.