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The prokaryotic transcriptome is shaped by transcriptional and posttranscriptional events that define the characteristics of an RNA, including transcript boundaries, the base modification status, and processing pathways to yield mature RNAs. Currently, a combination of several specialised short-read sequencing approaches and additional biochemical experiments are required to describe all transcriptomic features. In this study, we present native RNA sequencing of bacterial ( E. coli ) and archaeal ( H. volcanii, P. furiosus ) transcriptomes employing the Oxford Nanopore sequencing technology. Based on this approach, we could address multiple transcriptomic characteristics simultaneously with single-molecule resolution. Taking advantage of long RNA reads provided by the Nanopore platform, we could accurately (re-)annotate large transcriptional units and boundaries. Our analysis of transcription termination sites revealed that diverse termination mechanisms are in place in archaea. Moreover, we shed light on the poorly understood rRNA processing pathway in archaea and detected new processing intermediates. One of the key features of native RNA sequencing is that RNA modifications are retained. We could confirm this ability by analysing the well-known KsgA-dependent rRNA methylation sites. Notably, our analysis suggests that rRNA modifications are more abundant in a hyperthermophilic organism.

Pyrococcus furiosus DSM 3638 is a model organism for hyperthermophilic archaea with an optimal growth temperature near 100 degrees C. The genome was sequenced about 18 years ago. However, some publications suggest that in contrast to other Pyrococcus species, the genome of P. furiosus DSM 3638 is prone to genomic rearrangements. Therefore, we re-sequenced the genome using third generation sequencing techniques. The new de novo assembled genome is 1,889,914 bp in size and exhibits high sequence identity to the published sequence. However, two major deviations were detected: (1) The genome is 18,342 bp smaller than the NCBI reference genome due to a recently described deletion. (2) The region between PF0349 and PF0388 is inverted most likely due an assembly problem for the original sequence. In addition, numerous minor variations, ranging from single nucleotide exchanges, deletions or insertions were identified. The total number of insertion sequence (IS) elements is also reduced from 30 to 24 in the new sequence. Re-sequencing of a 2-year-old "lab culture" using Nanopore sequencing confirmed the overall stability of the P furiosus DSM 3638 genome even under normal lab conditions without taking any special care. To improve genome annotation, the updated DNA sequence was combined with an RNA sequencing approach. Here, RNAs from eight different growth conditions were pooled to increase the number of detected transcripts. Furthermore, a differential RNA-Seq approach was employed for the identification of transcription start sites (TSSs). In total, 2515 TSSs were detected and classified into 834 primary (pTSS), 797 antisense (aTSS), 739 internal and 145 secondary TSSs. Our analysis of the upstream regions revealed a well conserved archaeal promoter structure. Interrogation of the distances between pTSSs and aTSSs revealed a significant number of antisense transcripts, which are a result of bidirectional transcription from the same TATA box. This mechanism of antisense transcript production could be further confirmed by in vitro transcription experiments. We assume that bidirectional transcription gives rise to non-functional antisense RNAs and that this is a widespread phenomenon in archaea due to the architecture of the TATA element and the symmetric structure of the TATA-binding protein.

Transcription initiation by archaeal RNA polymerase (RNAP) and eukaryotic RNAP II requires the general transcription factor (TF) B/ IIB. Structural analyses of eukaryotic transcription initiation complexes locate the B-reader domain of TFIIB in close proximity to the active site of RNAP II. Here, we present the first crosslinking mapping data that describe the dynamic transitions of an archaeal TFB to provide evidence for structural rearrangements within the transcription complex during transition from initiation to early elongation phase of transcription. Using a highly specific UV-inducible crosslinking system based on the unnatural amino acid para-benzoyl-phenylalanine allowed us to analyze contacts of the Pyrococcus furiosus TFB B-reader domain with site-specific radiolabeled DNA templates in preinitiation and initially transcribing complexes. Crosslink reactions at different initiation steps demonstrate interactions of TFB with DNA at registers +6 to +14, and reduced contacts at +15, with structural transitions of the B-reader domain detected at register +10. Our data suggest that the B-reader domain of TFB interacts with nascent RNA at register +6 and +8 and it is displaced from the transcribed-strand during the transition from +9 to +10, followed by the collapse of the transcription bubble and release of TFB from register +15 onwards.

Transcription factor B recruiting factor 1 (TFB-RF1; PF1088) is a transcription regulator which activates transcription on archaeal promoters containing weak TFB recognition elements (BRE) by recruiting TFB to the promoter. The mechanism of activation is described in detail, but nothing is known about the biological function of this protein in Pyrococcus furiosus. The protein is located in an operon structure together with the hypothetical gene pf1089 and western blot as well as end-point RT-PCR experiments revealed an extremely low expression rate of both proteins. Furthermore, conditions to induce the expression of the operon are not known. By introducing an additional copy of tfb-RF1 using a Pyrococcus shuttle vector we could circumvent the lacking expression of both proteins under standard growth conditions as indicated by western blot as well as end-point RT-PCR experiments. A ChIP-seq experiment revealed an additional binding site of TFB-RF1 in the upstream region of the pf1011/1012 operon, beside the expected target of the pf1089/tfb-RF1 region. This operon codes for a putative ABC transporter which is most-related to a multidrug export system and in vitro analysis using gel shift assays, DNase I footprinting and in vitro transcription confirmed the activator function of TFB-RF1 on the corresponding promoter. These findings are also in agreement with in vivo data, as RT-qPCR experiments also indicate transcriptional activation of both operons. Taken together, the overexpression strategy of tfb-RF1 enabled the identification of an additional operon of the TFB-RF1 regulon which indicates a transport-related function and provides a promising starting position to decipher the physiological function of the TFB-RF1 gene regulatory network in P. furiosus.

EMSAs with TrmBL1 and 15 identified binding regions. (PDF 892Â kb)

In vitro transcription assays. (PDF 978Â kb)

Fragmentation of P. furiosus genomic DNA by sonication. (PDF 1040Â kb)

Transcription in Archaea relies on a eukaryotic-like transcription machinery composed of general transcription factors, the RNAP and corresponding promoter elements. In contrast, the regulation of archaeal gene expression depends on bacterial-like transcription factors. In the present work the gene regulatory networks of several archaeal transcription factors from the hyperthermophilic euryarchaeon P. furiosus were examined by ChIP. Moreover, combining ChIP with high-throughput sequencing (ChIP-seq) enabled the first time genome-wide TF binding site mapping in P. furiosus. ChIP-seq revealed that the global regulator of sugar metabolism in P. furiosus, TrmBL1, not only represses and activates expression of sugar-uptake-, glycolysis- and gluconeogenesis-specific-enzymes encoding genes as proposed, but it also controls transcription of genes encoding proteins catalyzing proteolysis, AA metabolism and nucleotide degradation. In addition, TrmBL1 indirectly affects expression of genes involved in functionally-linked metabolic pathways by transcriptional repression of the hitherto not well-characterized secondary transcription factor PF1476. In contrast, the anti-TrmB antibodies were not specific enough to allow investigation of the proposed co-regulation of TrmB and TrmBL1 in sugar metabolism. Furthermore, the genes encoding TrmB and the TM-system were deleted in the P. furiosus type strain. After successfull establishment of the ChIP-seq approach, genome-wide TF binding site mapping was extended to analyze a third paralogous TrmB protein designated as TrmBL2. These studies uncovered numerous potential functions of the TF as both architectural abundant DNA binding protein and global transcriptional regulator in P. furiosus. Furthermore, TrmBL2 ChIP-seq analysis using P. furiosus cells incubated at 107° for 30 min revealed a novel unexpected role of the protein in the heat shock response. TFB-RF1 functions as transcriptional activator by recruitment of TFB to an imperfect BRE. Under optimal growth conditions expression of TFB-RF1 is not detectable and the transcription factor had to be over expressed from the fbpase promoter. First combination of ChIP-seq with the recently developed in vivo over-expression system of P. furiosus confirmed binding of the TF to its in vitro designated binding site in vivo. However, no additional binding sites were identified, which would elucidate a physiological function as regulator. The presented studies of this work contribute to a better understanding of the mechanisms of transcriptional control mediated by gene regulatory networks to achieve optimal growth of the cells in response to differing environmental as well as intracellular conditions., Die Transkription in Archaeen beruht auf einem dem eukaryotischem System ähnlichen Transkriptionsapparat bestehend aus allgemeinen Transkriptionsfaktoren, der RNA Polymerase und den entsprechenden Promotorelementen. Im Gegensatz dazu wird die archaeelle Genexpression vor allem von Transkriptionsfaktoren reguliert, die dem bakteriellen System ähnlich sind. In dieser Studie wurden die genregulatorischen Netzwerke von mehreren archaeelen Transkriptionsfaktoren aus dem hyperthermophilen Modelorganismus P. furiosus durch Etablierung von ChIP Experimenten untersucht. Die Kombination von ChIP Experimenten mit Hochdurchsatz-Sequenzierung (ChIP-Seq) ermöglichte zum ersten Mal die Identifizierung von Bindestellen des jeweiligen Transkriptionsfaktors im gesamten Genom von P. furiosus. Im ersten Teil der Arbeit konnte durch ChIP-Seq nachgewiesen werden, dass der globale Regulator des P. furiosus Zuckerstoffwechsels, TrmBL1, nicht nur wie bekannt die Expression von Genen reprimiert und aktiviert, die Zuckeraufnahme-, Glykolyse und Glukoneogenese-spezifische Enzyme kodieren, sondern auch von Genen, die Proteine kodieren, die an der Proteolyse, dem Aminosäurestoffechsel und der Degradierung von Nukleotiden beteiligt sind. Zudem beeinflusst TrmBL1 durch Repremierung der Genexpression des bis jetzt noch nicht intensiv charakterisierten Regulators PF1476 die Transkription von Genen, die an funktionell-verbundenenen metabolischen Prozessen beteiligt sind. Eine Analyse der vermuteten Ko-Regulation des Zuckerstoffwechsels durch die zwei paralogen P. furiosus TrmB Proteine TrmB und TrmBL1 konnte sowohl aufgrund der Kreuzreaktivität der TrmB spezifischen Antikörper mit TrmBL1 als auch aufgrund der genomischen Deletion des Genes, das für TrmB kodiert, nicht durchgeführt werden. Im zweiten Teil der Arbeit wurden die ChIP-Seq Experimente auf eine Untersuchung der Funktion eines dritten paralogen TrmB Proteins, TrmBL2, ausgeweitet. Diese Versuche deckten zahlreiche potentielle Funktionen dieses Transkriptionsfaktors als sowohl strukturgebendes chromosomales DNA Bindeprotein als auch als globaler Transkriptions-regulator auf. Zudem wurde eine bis dato unbekannte und unerwartete Beteiligung von TrmBL2 an der Hitzeschockantwort von P. furiosus gefunden. Zusätzlich ermöglichte die erstmalige Kombination von ChIP-Seq Experimenten mit dem kürzlich entwickelten in vivo Überexpressionssystem in P. furiosus eine Charakterisierung von TFB-RF1 in vivo. Dieser fungiert als Aktivator der Transkription durch Stimulierung der Rekrutierung von TFB zum Promotor-TBP-Komplex. Unter normalen Wachstums-bedingungen ist TFB-RF1 nur schwach bzw. gar nicht exprimiert, was eine Induktion der Expression erforderte. Durch die Kombination beider Ansätze konnte die Bindung von TFB-RF1 an die in vitro bestimmte Bindestelle im Genom in vivo bestätigt werden. Es konnten jedoch keine weiteren Bindestellen nachgewiesen werden, die zur Aufklärung der physiologischen Funktion dieses außergewöhnlich archaeellen Aktivators beitragen könnten. Die in dieser Arbeit vorgestellten Ergebnisse tragen zu einem besseren Verständnis der durch genregulatorische Netzwerke vermittelten Transkriptionsregulation bei, die ein optimales Zellwachstum unter sich verändernden Umweltbedingungen ermöglicht.