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Archaea are a key feature of the terran biosphere. Since their early characterization in the 1970s, we have learned much about the fascinating organism, most recently by applying genome-, transcriptome-, proteome-, and metabolome-scale methods. This chapter describes seminal contributions that elaborate on the study of archaeal biology at the systems level.

Genome-wide occupancy studies for RNA polymerases and their basal transcription factors deliver information about transcription dynamics and the recruitment of transcription elongation and termination factors in eukaryotes and prokaryotes. The primary method to determine genome-wide occupancies is chromatin immunoprecipitation combined with deep sequencing (ChIP-seq). Archaea possess a transcription machinery that is evolutionarily closer related to its eukaryotic counterpart but it operates in a prokaryotic cellular context. Studies on archaeal transcription brought insight into the evolution of transcription machineries and the universality of transcription mechanisms. Because of the limited resolution of ChIP-seq, the close spacing of promoters and transcription units found in archaeal genomes pose a challenge for ChIP-seq and the ensuing data analysis. The extreme growth temperature of many established archaeal model organisms necessitates further adaptations. This chapter describes a version of ChIP-seq adapted for the basal transcription machinery of thermophilic archaea and some modifications to the data analysis.

The fully recombinant Methanocaldococcus jannaschii RNA polymerase allows for a detailed dissection of the different stages of the transcription. In the previous chapter, we discussed how to purify the different components of the M. jannaschii transcription system, the RNA polymerase subunits, and general transcription factors and how to assemble a functional M. jannaschii enzyme. Standard in vitro transcription assays can be used to examine the different stages of transcription. In this chapter, we describe how some of these assays have been optimized for M. jannaschii RNA polymerase, which transcribes at much higher temperatures than many other transcription complexes.

Archaeal RNA polymerase and general transcription factors are more closely related to those of eukaryotes than of bacteria. As such the study of transcription of archaea is important both in terms of examination of the evolution of the transcriptional machinery and as a simplified tool for eukaryotic transcription. In particular, the hyperthermophilic Methanocaldococcus jannaschii provides us with a fully recombinant RNA polymerase system allowing for much more detailed in vitro examination of the roles of different components during the transcription cycle than otherwise possible. The individual subunits of M. jannaschii enzyme are easily expressed and purified from heterologous expression systems. Forming functional RNA polymerase involves simply combining the different subunits under denaturing conditions and slowly removing the denaturant.

TFIIB-like general transcription factors are required for transcription initiation by all eukaryotic and archaeal RNA polymerases (RNAPs). TFIIB facilitates both recruitment and post-recruitment steps of initiation; in particular, TFIIB stimulates abortive initiation. X-ray crystallography of TFIIB-RNAP II complexes shows that the TFIIB linker region penetrates the RNAP active center, yet the impact of this arrangement on RNAP activity and underlying mechanisms remains elusive. Promoter-independent abortive initiation assays exploit the intrinsic ability of RNAP enzymes to initiate transcription from nicked DNA templates and record the formation of the first phosphodiester bonds. These assays can be used to measure the effect of transcription factors such as TFIIB and RNAP mutations on abortive transcription.