We identified a novel regulator, Thermococcales glycolytic regulator (Tgr), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a transcriptional repressor regulating the trehalose/maltose ATP-binding cassette transporter genes, but is more closely related (67%) to a TrmB paralog in P. furiosus (PF0124). Growth of a tgr disruption strain (⌬tgr) displayed a significant decrease in growth rate under gluconeogenic conditions compared with the wild-type strain, whereas comparable growth rates were observed under glycolytic conditions. A whole genome microarray analysis revealed that transcript levels of almost all genes related to glycolysis and maltodextrin metabolism were at relatively high levels in the ⌬tgr mutant even under gluconeogenic conditions. The ⌬tgr mutant also displayed defects in the transcriptional activation of gluconeogenic genes under these conditions, indicating that Tgr functions as both an activator and a repressor. Genes regulated by Tgr contain a previously identified sequence motif, the Thermococcales glycolytic motif (TGM). The TGM was positioned upstream of the Transcription factor B-responsive element (BRE)/TATA sequence in gluconeogenic promoters and downstream of it in glycolytic promoters. Electrophoretic mobility shift assay indicated that recombinant Tgr protein specifically binds to promoter regions containing a TGM. Tgr was released from the DNA when maltotriose was added, suggesting that this sugar is most likely the physiological effector. Our results strongly suggest that Tgr is a global transcriptional regulator that simultaneously controls, in response to sugar availability, both glycolytic and gluconeogenic metabolism in T. kodakaraensis via its direct binding to the TGM.Proper control of glycolytic and gluconeogenic activities in the cell is vital for the efficient assimilation of carbon and generation of energy and has been considered a paradigm for metabolic regulation. Stringent regulation is generally observed to avoid futile cycles that potentially lead to the depletion of energy; hence one pathway is suppressed while the other is active.Mechanisms underlying the regulation of glycolysis/gluconeogenesis have been extensively studied in different bacterial and eukaryotic species. In Escherichia coli, genes involved in these pathways are generally expressed in a constitutive manner (1), and control is brought about predominantly by allosteric regulation of the enzymes themselves. The major sites of allosteric regulation are the two glycolytic enzymes, phosphofructokinase (PFK) 3 (2) and pyruvate kinase (3), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) (4). The reactions catalyzed by the three enzymes are irreversible under physiological conditions and are therefore considered key steps in the respective pathways. Besides the allosteric control, recent analyses have also indicated the p...

Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and β subunits. Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEβ and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/β and its engagement with the RNAP clamp. TFEα/β stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the β subunit and the promoter sequence. Our results suggest that archaeal TFEα/β is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes.DOI: http://dx.doi.org/10.7554/eLife.08378.001

Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best in silico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be a universal mode of control in bacteria and archaea that allows for differential production of operon-encoded proteins.

14The archaeal transcription apparatus is closely related to the eukaryotic RNA 15polymerase ( that Spt4/5 is a general elongation factor in archaea since its presence on all 27 genes matches RNAP. Spt4/5 is recruited proximal to the TSS on the majority 28 of transcription units, while on a subset of genes including rRNA and CRISPR 29 loci, Spt4/5 is recruited to the transcription elongation complex during early 30 elongation within 500 bp of the TSS, and akin to its bacterial homolog NusG. 31 32